Mole Cell Exam #3

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The "zinc-finger" of transcription factors is an example of an ________ protein structural fold:

"alpha plus beta"

(+) ssRNA animal virus replication (e.g., HCV) - Step 2:

(+) ssRNA serves as the template for complementary (-) strand synthesis - viral proteins: proteins include RNA dependent RNA polymerase

(+) ssRNA animal virus replication (e.g., HCV) - Step 3:

(-) ssRNA serves as the template for complementary (+) strand synthesis - results in (+) ssRNA --> more viral proteins --> assembly into nucleocapsid

Initiation of Prokaryotes - The correct binding of the fmet-tRNA^fmet to the P site in the 70S initiation complex is ensured by (1) i. The codon-anticodon interaction involving initiation

(1) AUG fixed in the P site

What is the third step in the life cycle of a typical virus?

(Uncoating and) replication of viral genome

Anaphase

- Centromeres split - Sister chromatids separate

Prophase

- Chromosome condensation - Centrosome migration - Mitotic spindle assembles

Metaphase:

- Chromosomes move to spindle equator and are aligned at the metaphase plate

Inhibitors: Ricin Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Eukaryotes - Inactivates 60S subunit of ribosome

What are types of lipid additions?

- N-myristoylation - Prenylation - Palmitoylation

Hallmarks of cancer • Disregard normal cell growth control

- Self-sufficiency in growth signals - Insensitivity to growth inhibitory signals

Elongation in prokaryotes requires:

- The initiation complex - Aminoacyl-tRNAs - Elongation factors: EF-Tu, EF-Ts and EF-G - GTP

Hepatitis B (HBV, DNA virus integrates into liver cell genome):

- jaundice / darkening of urine - chronic hepatitis leading to liver failure

Hepatitis C (HCV, RNA virus):

- long-term infection --> chronic liver disease - most common reason for liver transplantation in the USA

Epstein Barr Virus (EBV) - LMP-1 - JAK/STAT signaling pathways [proliferation]; activates Bcl2 [inhibits apoptosis]

- mimics activated CD40 receptor - leads to activation of NF-κB;

M phase - Mitosis: ?? - Cytokinesis - spilt into 2 cells

- prophase - prometaphase - metaphase - anaphase - telophase

Mutagenic inactivation of tumor suppressor genes

-Loss of function -Mutations of both alleles are required

What is the lifecycle of a typical virus?

1. Attachment (adsorption) of virion to the correct host cell 2. Entry (penetration) of the virion or viral genome 3. (Uncoating and) replication of viral genome 4. Synthesis of viral proteins 5. Assembly of new virions 6. Release of new virions from the host cell

What are the four steps of prokaryotic gene transcription (no nucleus)?

1. Promoter recognition by σ factor of RNA polymerase holoenzyme) 2. Initiation 3. Elongation 4. Termination

RNA is synthesized in

5' --> 3' direction

In prokaryotes, tRNA^fmet is used when

5'AUG is the initiation codon

Underactivity mutation (loss of function)

A normal cell undergoes a mutation event that *inactivates tumor suppressor genes* then undergoes a *second mutation event* which inactivates the second gene copy. These two inactivating mutations *eliminate the tumor suppressor genes* and promote cell transformation

Elongation Step 2 in prokaryotes: peptide bond formation - fMet is transferred from its tRNA at the P site to the amino acid at the

A site

Human T-cell Leukemia Virus - Tax protein - also stimulates

AKT signaling pathway, activates NF-κB and cyclin D2, inhibits expression of CDK inhibitors

BRCA1 is a hotspot for

ALU

Chromosome Segregation - When M-Cdk becomes activated, it phosphorylates

APC

How are polypeptides transported?

Across the mitochondrial membrane

Cdc25A phosphatase

Activates vertebrate S phase CDKs

Positive regulation by cAMP "second messenger" - Bacteria sense lack of glucose metabolically and activate

Adenylyl cyclase enzyme

Caspases = A family of cysteine proteases that cleave after

Asp residues

Pro-apoptotic effector Bcl-2 family proteins, such as ____ and ____ are inactivated by interaction with anti-apopototic Bcl-2 family members.

Bax ; Bak

• There are more than 20 members in the Bcl-2 family - Pro-apoptotic Bcl-2 family proteins:

Bax, Bak, Bid, Bim

Bacterial - IF3

Binds to 30S subunit; prevents premature association of 50S subunit

Eukaryotic Transcription of rRNA by RNA polymerase I - Bipartite promoter for RNA pol I has an upstream control element and a core promoter rich in

C/G pairs

Initiator caspases contain a long pro-domain • Death effector domain (DED):

Caspase-8

Unreplicated or single-stranded DNA activates ATR which activates

Chk1 kinase

ATM is activated by double strand breaks and activates

Chk2

Properties of the genetic code - Non-overlapping:

Codons are read in a successive, non-overlapping fashion.

Eukaryotic - elF4F

Complex consisting of eIF4E, eIF4A, and eIF4G

Chromosome condensation - __________ and _____ _______ of chromatin (SMC) proteins are involved.

Condensins ; structural maintenance

Caspases =

Cysteine Aspartases

Mitochondrial or intrinsic cell death pathway • _________ released into the cytoplasm initiates apoptosis

Cytochrome c

Most human cancer viruses are _____ viruses.

DNA

Elongation of mRNA chain: Two proteins: ___ (DRB-sensitivity inducing factor) & _____ (negative elongation factor) bind RNA pol II and pause gene transcription after transcript has been initiated.

DSIF;NELF

Ubiquitin-Protein Ligases: APC/C^Cdh1

Degradation of B-type cyclins in G1 and geminin in metazoans to allow loading of replicative helicases on DNA replication origins

Oncogenic Viruses: RNA (HTLV-1) or DNA (HPV, EBV, HBV) Mechanisms?

Disrupt regulation of host cell proliferation (inhibit p53, pRb); Inhibit apoptosis; Activate growth signaling

Cytokinesis

Division of the cytoplasm during cell division

Initiation in Eukaryotes Step 1: - (3) elF1 binds to the

E site

During the process of protein synthesis, the factor eEF-2 induces the hydrolysis of GTP. The energy of this hydrolysis is coupled to which of the following A. Amino acid activation by attachment to a tRNA B. Correct alignment of the mRNA on the 40S ribosome. C. Formation of the 80S initiation complex. D. Formation of the peptide bond E. Translocation of the ribosome

Proteins that do not fold properly in the endoplasmic reticulum (ER) lumen are degraded in the cytosol by the A. nucleases B. microsome C. Ribosome D. Endosome E. Proteasome

Human Papilloma Virus (HPV) - E6/E7 regulated by

Passage through the restriction point - Phosphorylated Rb cannot bind to _____.

E2F

The transcription of cyclin E and cyclin A is dependent on

E2F transcription factor

Role of TGM2 in TGFβ2-OHT - TGFβ2 ECM fibrosis in glaucoma • Includes ECM cross-linking • TGM2 = ????

ECM cross-linking enzyme

Fibrotic Diseases Often Have Increased Expression of the

EDA-Fibronectin Isoform

Termination in Prokaryotes • Termination is triggered by the stop codons UAA, UAG and UGA at the A site. • The release factors (RFs) bind to the termination codon at the A site. • Release factors hydrolyze the terminal peptidyl-tRNA bond. • The complete peptide is released from the ribosome. • The uncharged tRNA, mRNA, and RFs leave the ribosome. • The ribosome recycling factor (RRF) and the hydrolysis of ___ bound GTP aid dissociation of ribosome subunits.

EF-G

Proto-oncogenes serve important cellular functions - Growth Factors

EGF, PDGF

Properties of the genetic code - Unambiguous

Each codon specifies only one amino acid

Explain the 4 stages of RNA transcription in prokaryotes: - Stage 3

Elongation of RNA

_____ is amplified in breast cancer

ErbB2

G1/S cyclins:

Expressed late in G1

Prokaryotes _________: A complex of ribosomes and RNA polymerase that begins translation during transcription.

Expressome

Carboxylation

Extra carboxyl groups may be added to Glu residues.

Inhibitor: Cip/Kip family (p21, p27, p57) Cdk or Cdk/cyclin complex: Cdk2/cyclin E Cell cycle phase inhibited: ???

Inhibitor: Ink4 family (p15, p16, p18, p19) Cdk or Cdk/cyclin complex: Cdk4 and Cdk6 Cell cycle phase inhibited: ???

What are the CDK-4 and CDK-6 (cyclin D) function?

G1 - entry into the cell cycle

Interphase is

G1 phase, S phase, G2 phase

If preferred glucose is present, less favored sugars not used. When glucose runs out, other sugars must be consumed --->

Genes (and encoded proteins)

• Point mutation in Ras is the most common abnormality in proto-oncogenes in human. • The frequency of mutation varies with the tumor type. • A single nucleotide change, which alters codon 12 from _______ can contribute to its transforming activity.

Gly to Val

Epstein-Barr virus (EBV) - Type: DNA - Family: _______ - Cancers caused: Burkitt's lymphoma, nasopharyngeal carcinoma

Herpesvirus

Organization of prokaryotic promoters -10 box:

Highly conserved 6 base sequence on DNA coding strand.

Eukaryotic - elF1A

Homolog of bacterial IF1; prevents premature binding of tRNAs to A site

Laminopathies: Defective LMNA Spicing can cause a variety of disease including

Hutchison-Gilford progeria syndrome (HGPS)

Initiation of Eukaryotes - eIF1A and eIF3 are equivalent to

IF-1 and IF-3

S cyclins:

Induced at late G1 after cyclin E

G1 cyclins:

Induced by growth factors or mitogens early in G1 phase

Ink4

Inhibits cdk4

Frame shift mutation:

Insertion or deletion of nucleotides (not a multiple of three) within the coding region of a gene. (the reading frame is altered)

Enhancer insertion:

Integrate far away from proto-oncogene; LTRs stimulate expression ("enhancer effect")

Classification of viruses - On the basis of viral genome material.....

It can have DNA or RNA, circular or linear, double-stranded or single stranded.

A 66 year old man was diagnosed with bladder carcinoma. A biopsy identified a single point mutation which resulted in the inabililty to hydrolyze GTP (guanosone-5-triphosphate), to GDP (Guanosine-5'-diphosphate). Which of the following oncogenes is most likely implicated in this case?

K-RAS

Kip1

Kinase inhibitory protein 1

p53 - Inherited for genetic transmission of a rare hereditary cancer syndrome called

Li-Fraumeni syndrome

What is the shortest phase in cell cycle?

M phase (most complex)

Activator Proteins and the "______" regulate eukaryotic gene transcription at a distance

Mediator

Proto-oncogenes serve important cellular functions - Transcription factors:

Myc

Caspases are synthesized as inactive proenzymes that contain 3 domains:

NH2 terminal pro-domain, large subunit and small subunit.

Properties of the genetic code - ______ ______ through prokaryotes, eukaryotes and plant kingdom

Nearly universal

Tauopathies:

Neurodegenerative diseases with pathological aggregation of hyperphosphorylated tau in neurons and/or glia

Conversion of proto-oncogenes to oncogenes - Gene Amplification:

Numerous copies of a protooncogene leads to overproduction of the encoded protein

e.g., Octamer element recognized by separate transcription factors ____ and _____:

Oct-1; Oct-2

- ________ join to the inositol head group of phosphatidylinositol - The two fatty acid chains of the lipid are embedded in the plasma membrane.

Oligosaccharides

Palmitoylation

Palmitic acid is added to an internal Cys residue.

_____ regulation of the lac operon by cAMP "second messenger"

Positive

Prenylatoin

Prenyl groups are attached to the side chains of Cys residues.

Proteolytic processing Protein maturation: • Proteolytic cleavage converts inactive precursor proteins to active forms • Ex:

Proinsulin, proteases (chymotrypsinogen, trypsinogen), viral proteins (HIV).

Eukaryotic - elF5 elF5b

Promotes dissociation of several other initiation factors from 40S subunit. GTPase homologous to bacterial IF2; promotes dissociation of initiation factors before final ribosome assembly

______ Analysis Experiment Visualizes Introns

R-Loop

Antisense strand is template strand transcribed to produce _____.

RNA

What is the sixth step in the life cycle of a typical virus?

Release of new virions from the host cell

G1 Cyclins - Cyclin D

Required for entry into the cell cycle.

_______ initiate protein synthesis in the cytosol.

Ribosomes

Activation of Cyclin E-Cdk2 - Cyclin E-Cdk2 becomes fully activated and cells enter the

S phase

Passage through the restriction point - E2F transcribes genes necessary for

S phase transition

Inhibitor: Cip/Kip family (p21, p27, p57) Cdk or Cdk/cyclin complex: Cdk2/cyclin A Cell cycle phase inhibited: ???

S, G2

Pre-mRNA Splicing and Pathogenesis of Human Diseases - Another example is the Ron (MST1R) proto-oncogene. An important property of cancerous cells is their ability to move and invade normal tissue - Production of an abnormally spliced transcript of Ron has been found to be associated with increased levels of the _____ in breast cancer cells - The abnomal isoform of the Ron protein encoded by this mRNA leads to cell motility

SF2/ASF

Discovery of New Glaucoma Pathogenic Pathway • Compare gene or protein expression between tissues or cells derived from glaucoma or control donor eyes • Increased expression of _____ in glaucoma cells and increased ____ in aqueous humor from glaucoma patients • Both ___ and ___ are inhibitors of the Wnt signaling pathway • How do we prove that these proteins are involved in glaucoma? - only have an association

SFRP1;DKK1

What are inhibitors of Wnt signaling pathway in Glaucoma?

SFRP1;DKK1

TRANSPOSONS ARE

SIMPLE DNA ELEMENTS

Eukaryotic Transcription of rRNA by RNA polymerase I • Subsequently, ____ binds to the DNA in association with UBF1

SL1

e.g., GC box (GGGCGG) seen in multiple copies. Although non-symmetrical, GC box works in either orientation --> bound by

SP1 transcription factor.

Targeting proteins to endoplasmic reticulum - Step 3:

SRP, nascent polypeptide and ribosome complex binds to GTP-bound SRP receptor in the ER membrane.

The genetic code is said to be degenerate because:

Several different codons may specify the same amino acid.

Processed Pre-mRNA in the Nuclear Compartment Associates with _______ _____ Proteins Before Transport

Specific hnRNP

Processed Pre-mRNA in the Nuclear Compartment Associates with __________ Before Transport

Specific hnRNP Proteins

(T or F): Transposons are not limited to eukaryotic organisms. They are found in most all prokarytoes and eukaryotes (originally identified in maze (corn))

Eukaryotic Transcription of rRNA by RNA polymerase I - Finally, RNA pol I binds with help from general transcription factor ____, and transcription commences

TIF1A

Explain the 4 stages of RNA transcription in prokaryotes: - Stage 4

Termination: Rho (ρ)-independent and ρ-dependent

Initiation in Eukaryotes Step 4:

The 48S complex scans the mRNA until it finds the AUG initiation codon in the conserved Kozak sequence.

Lipidation

The covalent attachment of lipid moieties to a proteins.

Targeting proteins to endoplasmic reticulum - Step 8:

The nascent protein is drawn into the ER lumen. Translocon closes.

Targeting proteins to endoplasmic reticulum - Step 2:

The signal sequence and the ribosome bind to a large complex called signal recognition particle (SRP). SRP binds to GTP and halts protein synthesis.

Secretory Pathway -

The synthesis of proteins destined for endoplasmic reticulum (ER), secretory pathway, lysosomes and membranes are initiated in the cytoplasm but ends after co-translational targeting to ER. Proteins may be further sorted in the Golgi.

Targeting proteins to endoplasmic reticulum - Step 7:

The translation completes at mRNA stop codon. The ribosome dissociates.

In humans, transposons elements are generally inactive. Transposons can move via two different mechanisms. • Conservative transposition - ?? • This does not increase the number of transposons in the DNA. • It does create double stranded cuts in the DNA and activates the double stranded DNA repair mechanism. This can be detrimental to the cell. • Therefore, there is a high selection pressure not to transpose.

The transposon is excised from the DNA and then re-inserted elsewhere.

Gene Duplications - Creation of pseudogenes • Unequal recombination: Gene is duplicated based on homologous or non-homologous recombination

These pseudogenes also have introns and exons; they may or may not have promoter sequences attached to them

Rifamycins Mode of action resistance?

They can mutate the rpoB gene (encodes beta-subunit of RNA polymerase)

Cdc25 phosphatase dephosphorylates

Thr-14 & Tyr-15

CDK-activating kinase (CAK) phosphorylates at ________

Thr-161

- ________ of nucleotide sequence of a gene, through the medium of mRNA into the amino acid sequence of a protein - 300 macromolecules participate - 90% of the chemical energy used by a cell for all biosynthetic reactions is invested in protein synthesis

Translation

_____ Trans-esterification Reactions Take Place During Splicing of Two Exons

Two

Helix-loop-helix

Two alpha-helical regions linked by a loop of variable length. Common in proteins involved in multi-cellular eukaryotic development pathways.

Sporadic cases of retinoblastoma require

Two somatic inactivating mutations, one in each of the two copies of Rb

Sense strand of DNA has the same direction (5'-3') and sequence of transcribed RNA except T is replaced by

U in RNA

• A loop forms by association of U1 and U2 • U6 displaces ____ from the spliceosome and U4 departs as well

Ubiquitin-proteasome pathway Step 1:

Ubiquitin is activated by the enzyme E1.

Ubiquitin-proteasome pathway Step 3:

Ubiquitin ligase E3 associates with both E2 and a target protein and catalyzes the transfer of ubiquitin to a specific target protein.

Eukaryotic promoters are bipartite:

Upstream control elements + Core promoter

Retrovirus replication: - Step 4:

Viral mRNA exits nucleus, translated into viral proteins, and packaged with viral RNA genome and viral reverse transcriptase into new virions.

What is the replication of double stranded RNA (dsRNA)?

Virus contains RNA-dependent RNA polymerase, (+) strand used both as a template for (-) strand and as template for translation ("as an mRNA")

The life cycle of a virus 5. Assembly of new virions 6. Release of new virions - Capsid components self-assemble and associate wxith nucleic acid to form nucleocapsid • Enveloped viruses?

Virus-specific glycoproteins are synthesized and incorporated into the host cell membrane --> nucleocapsids then bind these membrane proteins (with help from viral matrix proteins) --> mature virions created by "budding"

Environment? + Glucose , + Lactose

We have glucose and lactose. But because we have glucose present it does not matter if lactose is present because we are not going to waste our energy to make lactose. So signal is off.

overactivity mutation (gain of function)

When a normal cell undergoes a *single mutation event* that creates an *oncogene*. This activating mutation then enables the oncogenes to promote cell transformation

What is a good nuclease?

When a nuclease only hits the on-target and very little of the off-target

What is the most common DNA-binding motif in human genome?

Zinc fingers

autocrine growth stimulation

a cell produces a growth factor (themselves) and interacts with the receptor to stimulate proliferation.

Restriction point

a cell that passes this point is committed to pass into S phase

Capping reaction uses three separate enzymes: FIRST: phosphatase removes 5' phosphate from transcript Second:

a guanyl transferase adds GMP by 5' to 5' linkage

Once post transcriptional processing is complete, (ie. Capping, Polyadenylation, Exon splicing, intron removal and methylation), the mRNA message remains associated with hnRNP proteins in

a messenger ribonuclear protein complex, mRNP

Capping reaction uses three separate enzymes: FIRST: phosphatase removes 5' phosphate from transcript Second: a guanyl transferase adds GMP by 5' to 5' linkage THIRD:

a methyl transferase adds methyl group to guanosine base (and, often,to 2' hydroxyls of ribose sugars of bases 1 and 2...)

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA - exceptions do occur: e.g. genes encoding histone mRNAS do not receive

a poly(A) tail and are instead processed differently

Components of Lac operon Single lac mRNA is translated to produce: LacA --->

a transacetylase protein

Hydroxylation

addition of hydroxyl groups

5' Splice Site, 3' Splice Site, and Branch Site All Are Important for Splicing • This is shown in detail for the U1 SNURP which recognizes the 5′ splice site • In the middle of the intron is a special _____ residue used as a branch site during splicing

adenine

Rb is also involved in tumors of the

adults

Some of the protein misfolding diseases are associated with the ____ of misfolded proteins.

aggregation

RNA synthesis in prokaryotes - Single RNA polymerase α2ββ'ωσ (holo enzyme) and α2ββ'ω (core enzyme) synthesize

all 3 classes of RNAs in prokaryotes (bacteria or bacteriophages)

Oct-1 found in _______

all tissues

Other causes of Thalassemia • Point mutations in coding regions can alter ______ β-globin

amino acid sequence

Amino acid activation • The correct pairing of an amino acid residues with an appropriate tRNA is achieved by a family of enzymes called

aminoacyl-tRNA synthetases.

Proteolytic processing • Removal of N-terminal methionine by an _____________.

aminopeptidase

Specific proteins misfold to form fibrous aggregates called _____.

amyloids

The philadelphia translocation involves:

an exchange between chromosome 9 and 22

separation of sister chromatids

anaphase

- Shortly after transcription starts, 5′ end of growing RNA molecule is capped by adding a guanosine triphosphate (GTP) residue (in the nucleus). - GTP is added in a _____ orientation (5'-5' Phosphodiester bond) relative to rest of the ribonucleotides (3'-5' Phosphodiester bond) in the RNA. - _______ connection stabilizes mRNA --> protects against mRNA degradation by nucleases.

backward

Rifamycins Mode of Action: Inhibits bacterial DNA-dependent RNA synthesis by inhibiting ___________. Binds specifically to beta-subunit of RNA polymerase; Broad spectrum and good penetration into tissues.

bacterial DNA-dependent RNA polymerase

Eukaryotic promoters are _____:

bipartite

Finally, splicing proceeds in three steps • Third, the intron is released as a _______ _______ ______ that is later degraded.

branched lariat structure

Bcl-2 overexpression is found not only in lymphomas but also in solid tumors, such as

breast cancer

EGFR or HER family of growth factor receptor tyrosine kinases are overexpressed in several cancers. HER2 or erbB2 is overexpressed in

breast cancers

Once multiple viral copies are assembled, the host cell ______ to allow progeny to escape and find new cells to infect.

bursts

Chromosomal translocation - _______ encodes a transcription factor normally induced in response to growth factors

c-Myc

An inability to degrade proteins that promote cell division or too-rapid degradation of proteins that inhibit cell proliferation can cause ______.

cancer

Tumor viruses can cause cancer: - Transformed cells have higher growth rate and divide indefinitely resulting in

cancer

Oncogenes:

cancer causing genes

Characteristics of Viruses - "Subcellular parasites" that strictly rely on host cell for energy & raw materials - Virus particles ("virions") contain RNA or DNA genomes protected by a shell of protein ("_____").

capsid

The Bcl-2 family protein Bid is a substrate for

caspase-8

p53 acts as a transcription factor and induces genes involved in ___________ (e.g., p21), DNA repair (e.g., GADD45) and apoptosis (e.g., Bax)

cell cycle arrest

Autophagy is believed to be a cellular protective mechanism, extensive autophagy has been associated with

cell death

Bcl-2 was the first protein that was shown to contribute to cancer by inhibiting ____ ____ by inhibiting apoptosis.

cell death

Lytic - Infection results in host ____ ___. Virus inhibits host cells synthetic enzymes, resulting in ___ ____.

cell death

Retrovirus envelope is made from the ____ ____ of its previous host cell. This membrane layer has retrovirus proteins both inserted through it and covering its surface.

cell membrane

The sense strand is the

coding strand

CDK1 - which cyclins?

cyclin A / cyclin B

Rifamycins strongly induce _______, thereby increasing degradation/elimination of warfarin, glucocorticoids, opioid analgesics, oral anti-diabetic drugs, and estrogen (decreasing the effectiveness of oral contraceptives).

cytochrome P450 enzymes

Nuclear pore complexes control export of mRNA from the nucleus to ________.

cytoplasm

Unfolded proteins are transferred to chaperonins where they are shielded from the

cytosol

Chaperonins consist of multi subunits and form a

double-chambered structure

Gene Therapy - Genome Editing •Gene knockout (________)

eliminate

Entry (penetration) of the virion or viral genome - Variable "mechanics" depending on type of virus... - e.g., receptor-mediated ________ of influenza virus & release of genome from endosome:

endocytosis

RNA Interference • Small interfering RNA (siRNA):

endogenous or exogenous

3' pre-mRNA cleavage and polyadenylation also ____ DNA transcription by RNA pol II complex

ends

What are the CDK-2 (cyclin E & A) function?

entry into the cell cycle S phase

Entry (penetration) of the virion or viral genome. - Variable "mechanics" depending on type of virus... - e.g., merger of ______ layer with animal host cell membrane:

envelope

(-) ssRNA animal viruses (e.g., rabies, mumps, measles, influenza, Ebola) All (-) ssRNA virus are ______.

enveloped

Gene Therapy - DNA Targets •ncDNA

epigenetics

Gene Therapy - Viral Vectors • Genome integration vs

episomal (not integrated)

R-Loop Analysis Experiment Visualizes Introns • mRNA is hybridized to single-stranded DNA from the corresponding gene • Regions of base pairing (within the exons) are interrupted by loops due to the

extra intron sequences within the DNA

Once cells progress through the restriction point - Cells are committed to proceed through the S phase. - Cells lose their dependency on ________ factors.

extracellular

Cyclin E-Cdk2 and Cyclin A-Cdk2 can further phosphorylate Rb even when growth factors are removed, and D-type cyclin levels ____.

fall

Termination in prokaryotes: Both ρ-independent and ρ-dependent • Polymerase core α2ββ'ω recognizes stop sequence on template strand, stalls, then _____ DNA complex

falls off

CAP is performed _____ and tail is performed _______.

first;second

Environment? - Glucose , + Lactose

glucose is absent, hence Crp (+cAMP) binds, and lactose is present, hence the LacI repressor is removed. So the signal is on.

Most ______ are destined either for secretion or for incorporation into the plasma membrane.

glycoproteins

In the HIV genome, what does env encode?

gp160 (cleaved into gp120 & gp41)

D-type cyclins are the

growth factor sensor

Identified as an oncogene. However, Bcl‐2 did not affect cell proliferation, but promoted cell survival in

growth factor-deprived cells

Shortly after transcription starts, 5′ end of growing RNA molecule is capped by adding a _______ residue (in the nucleus).

guanosine triphosphate (GTP)

Classification of Viruses - On the basis of Structure: ____ _____ - Virus particles come in a wide range of sizes and shapes

helical or spherical

Positive regulation of prokaryotic gene transcription - Bound activator protein:

helps recruit RNA polymerase

The Ubiquitin-proteasome pathway is ___ ____.

highly conserved

Hallmarks of cancer - Escape

immune destruction

HDAC can

inhibit gene transcription

Most of the antibiotics inhibit protein synthesis in the elongation phase EXCEPT streptomycin which inhibits in the

initiation phase

Viruses in the bacterial (prokaryotic) world - Head structure contains DNA genome attached to a tail that contracts for

injecting the genome into the host

Transposons for gene therapy Sleeping Beauty transposon family can be used for gene therapy • First transposon found to be active in eukaryotes (a salmon transposon) • Currently being tested as a gene therapy agent for AMD • Useful because the transposase can be separated from the _____ _____, therefore once it is transposed into the gene, no worry that it will become an active transposon

insertion sequences

RNA tumor viruses - mechanisms?

insertional mutagenesis - promoter insertion - enhancer insertion

Amino acid activation - Two high-energy phosphate bonds are expended for each amino acid activation. Thus, the overall reaction of amino acid activation is essentially ______.

irreversible

Aggregation of polypeptides is prevented in the ______ environment

isolated

Finally, splicing proceeds in three steps • Second, the free 3′ end of the upstream exon displaces the intron from the 3′ splice site and the two exons are

joined together

The repressor in bacterial transposon is used to keep the transposon from

jumping

The metaphase spindle - Sister chromatids are attached to microtubules via _______.

kinetochore

The peptidyl transferase lies in the 60S that is also

part of the 80S in eukaryotes

Elongation Step 2 in prokaryotes:

peptide bond formation

What are types of mutations?

point mutation and frameshift mutation

Initiation in Eukaryotes Step 3: The elF4F complex binds to mRNA - eIF4F complex: - elF4G binds to

poly(A) binding protein (PABP)

Prokaryotic genes are ______. Several genes transcribed as single RNAs from same promoter/operator.

polycistronic

The HIV genome has protein products of gag, pol, and env are actually ______ that give rise to two or three proteins after cleavage

polyproteins

Prokaryotes - An mRNA bound to several ribosomes is called

polysomes or polyribosomes

Multiple ubiquitin molecules are attached to form a

polyubiquitin chain

Eukaryotic gene control region (for mRNA coding genes): • TATA box assembles RNA pol II and general transcription factors to form

pre-initiation complex

Cyclins are synthesized and degraded in a

precisely timed sequence

The primary transcript of eukaryotic gene transcription is a ______ or "Pre-RNA" (true for rRNA, mRNA, and tRNA genes)

precursor

Bacteria can grow on various sugars: fructose ("fruit sugar"), lactose ("milk sugar"), maltose (from starch breakdown), and glucose is

preferred

Most effective antibiotics act by inhibiting

protein synthesis

Eukaryotic pre-RNAs undergo processing to become mature, functional RNAs • Only mature RNAs (rRNA, mRNA, tRNA) participate in

protein translation in eukaryotes

Vitamin K-dependent carboxylation of Glu in _____ is involved in blood clotting.

prothrombin

Prokaryotes _____ RNA polymerase alone starts initiation / elongation of three RNA coding gene classes

single

Chaperones stabilize _____ polypeptides during translation or during transport of proteins to subcellular organelles.

unfolded

Cytosolic chaperones bind to and stabilize the .

unfolded configuration

What is the aggregating protein in Parkinson's Disease?

α-Synuclein

Why study transposons?

• Approximately 45-50% of the human genome is composed of transposable elements • Transposition can cause mutations and changes in gene regulation, which can cause disease

Gene Therapy - DNA Targets •Mutations

• Loss of function • Gain of function

RNA polymerase II for class II gene

(protein coding genes) to synthesize mRNA

RNA polymerase I for class I gene

(rDNA) transcription to synthesize rRNA

Overexpressed in 85% of patients with follicular B-cell lymphoma due to chromosomal translocation

(t14;18)

Eukaryotic gene control region (for mRNA coding genes): • enhancer elements can occur far upstream (or even far downstream) of

+1 site encompassing the TATA box.

(-)ssRNA animal viruses: Step 1: Synthesis of (+) ssRNA Step 2 ?

- (+) ssRNA is used to make more (-) ssRNA - (-)ssRNA is used to make (+) ssRNA ("mRNA") --> translated to proteins

N-linked

- (attached to Asn) • Takes place in the ER • The sugars joined to the amino acids are usually N-acetylglucosamine.

O-linked

- (attached to Ser or Thr) • Takes place in the Golgi • The sugars joined to the amino acid are usually N-acetylgalactosamine

autophagy - auto-: self and -phagy: to eat

- A process of self cannibalism by which a cell recycles its own components to survive under nutrient-deprived condition.

A ribosome contains 4 binding sites for RNA molecules: 1 for the mRNA and 3 for the tRNA

- A site: Aminoacyl-tRNA - P site: Peptidyl-tRNA - E site: Exit

What are diseases caused by protein aggregation?

- Alzheimer's - Parkinson's - Type 2 Diabetes

Point Mutation - Silent (no change):

- An alteration in codon that specifies the same amino acid. - Has no effect on protein - UCC --> UCA both code for Serine

Retrotransposons and disease In cancer cells, we can see disruption of genes by transposition

- BRCA1 in particular is a hotspot for ALU - ALU recombination and insertion - BCA1 has 137 ALU elements in it's introns

Type of Bacteriophage: Simple, small, single-stranded DNA viruses (ssDNA):

- Bacteriophage ΦX174 (has overlapping genes in genome); - Bacteriophage M13 has single-stranded circular DNA like ΦX174

Type of Bacteriophage: Complex double-stranded DNA viruses (dsDNA)

- Bacteriophages T4, Lambda (l), P1, and Mu (m) - Made up of a head, tail, and tail fibers.

Epstein Barr Virus (EBV) is implicated in several malignancies including

- Burkitt's lymphoma (more common in Africa) - B-cell lymphomas (in immunocompromised individuals) - Nasopharyngeal carcinomas (more common in Asia)

Contain dsDNA: Herpes simplex virus (HSV):

- Causes cold sores, genital herpes, as well as chickenpox (VZV). - Latent herpes viruses are found in the nucleus where they replicate as extra-chromosomal circular plasmids. - Active infections may break out again after a long period of quiescence, due to stress or other factors --> "Shingles"

Future Therapeutic Approaches •Treatment of epigenetic diseases •List some epigenetic diseases • How will epigenetic diseases be treated? • How will you selectively treat target cells or tissues?

- Colon cancer - Wnt signling suppressed due to expression of antagonists SFRP1 or DKK1. Methylation of SFRP1 allows unchecked Wnt signaling. - Crispr-cas9 can be used to methylate or demethylate to regulate the expression of genes that may have been advertent regulated in disease.

Regulation of cyclin-dependent kinases

- Cyclins - Phosphorylation-dephosphorylation - Cyclin-dependent kinase inhibitors (CKI) - Proteolysis - Transcription

What are diseases caused by protein misfolding?

- Cystic Fibrosis - α1-antitrypsin deficiency - Glucose-6-phosphate dehydrogenase (G6PD)

Telophase

- Daughter chromosomes arrive at the spindle poles - Nuclear membranes reform - Chromosomes decondense

Inhibitors: Diphtheria Toxin Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Eukaryotes - Inhibits eEF-2 and prevents translocation

Inhibitors: Cycloheximide Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Eukaryotes - Inhibits peptidyl transferase activity of 80S ribosome

Point Mutation - Nonsense:

- Formation of a stop codon. - Premature termination results in truncated protein - May be non-functional

What environment induces expression of the lac operon?

- Glucose , + Lactose

Activation of initiator caspases occurs via two distinct pathways:

- Intrinsic or mitochondrial pathway - Extrinsic or receptor-initiated pathway

Prometaphase

- Nuclear envelope breakdown - Chromosomes attach to spindle microtubules via kinetochores

Proteins undergo further modifications in the Golgi. - Examples?

- Oligosaccharies are O-linked to some proteins. - N-linked oligosaccharides are further modified.

Necrosis - "Nekrosis" means "deadness"

- Pathologic or accidental cell death - Cells swell and burst releasing their intracellular contents

Apoptosis - came from the Greek word "apo"-"away from" and "ptosis" to "drop off"

- Physiologic form of cell death - Genetically programmed and often referred to as programmed cell death (PCD)

Inhibitors: Erythromycin Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Prokaryotes - Binds to the 50S subunit and inhibits translocation

Inhibitors: Streptomycin Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Prokaryotes - Causes misreading of the genetic code. Interferes with initiation.

Inhibitors: Tetracycline Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Prokaryotes - Inhibits binding of aminoacyl-tRNA to the A-site

Inhibitors: Choramphenicol Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Prokaryotes - Inhibits peptidyl transferase activity on ribosomes

Inhibitors: Puromycin Prokaryotes, Eukaryotes, or Both? Mechanism of action: ??

- Prokaryotes and Eukaryotes - Resembles aminoacyl-tRNA and binds to A-site Causes premature release of polypeptide chain

Classification of Viruses: On the basis of viral envelope... - General Design Principles:

- Protein-containing lipid membrane around the nucleocapsid. - Lipid membrane is derived from host cell membrane "Viral package" contains genome + virus-specific proteins

Point Mutation - Missense

- Results in the incorporation of a different amino acid due to a single base change. - Possible change in the function of protein

Initiation of Prokaryotes - Initiation of polypeptide synthesis requires:

- The 30s ribosomal subunit - The mRNA coding for the polypeptide to be made - The initiating fMet-tRNA^fmet - A set of 3 proteins called initiation factors (IF) - IF-1, IF-2, and IF-2 - GTP - The 50s ribosomal subunit

Epstein Barr Virus (EBV) - EBNA-2 - Notch receptor activation

- activates cyclin-D and Src family kinases (proliferation)

(+) ssRNA animal viruses - Picornaviruses (non-enveloped)

- small, icosahedral - e.g., polio virus, common cold "rhinovirus", Hepatitis A, and cattle "foot and mouth" disease

replicative transposition

- target site is cleaved (ss break) - donor transposon is used to join cleaved target site - This forms a cointegrate with the donor DNA and the recipient molecule - New DNA is synthesized - Crossing over occurs at the internal resolution site

Bacteriophage treatment of patients with pulmonary infections

- those phages will detect and destroy the bacteria

Polymerase recognition of gene promoter in prokaryotes • Sigma factor (σ) or RNA polymerase holoenzyme (α2ββ'ωσ) binds to the _________ boxes of the prokaryotic gene promoter.

-10 and -35

Polymerase recognition of gene promoter in prokaryotes • RNA polymerase core enzyme (α2ββ'ω) is not efficient in binding to start site via ______ (TATAAT; "Pribnow box")

-10 box

Mechanisms of tumor suppression by p53:

-Activation of temporary cell cycle arrest (*quiescence*) -Activation of permanent cell cycle arrest (*senescence*) -Induction of *apoptosis*

Mutagenic activation of oncogenes

-Gain of function -Mutation of single allele is sufficient

Two types of enzymes catalyze protein folding:

-Protein disulfide isomerase (PDI) -Peptidyl proline isomerase

A ribosome contains 4 binding sites for RNA molecules:

1 for the mRNA and 3 for the tRNA

Sequence of events for activating eukaryotic gene transcription:

1. A specific transcription factor binds to DNA. 2. A histone acetyl transferase (HAT) binds to the specific transcription factor. 3. HAT acetylates histones in the vicinity and association of nucleosomes is loosened. 4. Chromatin-remodeling complex slides or rearranges the nucleosomes, allowing access to the DNA. 5. Further transcription factors bind. 6. RNA pol II and its general transcription factors bind to the DNA. 7. Initiation requires a positive signal to be transmitted via the mediator complex from the sum of the influence of one or more specific transcription factors.

What are the four steps of eukaryotic gene transcription (in nucleus)?

1. Formation of pre-initiation complex by general transcription factors at promoter's TATA box 2. Initiation 3. Elongation 4. Termination

Most specific transcription factors share these properties:

1. Respond to a stimulus that signalsa gene should be turned on / off. 2. Enter the nucleus where genes are. 3. Bind specific DNA sequences. 4. Make contact with the RNA pol II transcription apparatus (either directly or indirectly)

Activators/repressors contain DNA-binding and trans-activation/repressor domains • There are at least four types of DNA-binding regulatory proteins that bind DNA to regulate eukaryotic gene transcription. • These specific transcription factors contain one of the following DNA-binding domains:

1. Zinc fingers 2. Helix-turn-helix 3. Leucine zipper 4. Helix-loop-helix

Gene Therapy • Recent success in restoring night vision to LCA patients that were blind for decades • _______-fold improvement in light detection

10,000

Initiation of Eukaryotes - Requires at least ___ proteins, which are designated as eIFs (e.g., eIF-1, elF-2, etc.)

Oncogenic DNA Viruses - About____________ related to viral infections

12% of human cancers

BRCA1 has __________ elements in its intron

137 ALU

In prokaryotes, the Shine Dalgarno sequence is recognized by the pyrimidine-rich sequence near the 3' end of the

16s rRNA of the 30S ribosomal subunit.

Organization of prokaryotic promoters - Spacing also important:

17-19 base sequence and 5-9 base sequence before start site.

Eukaryotic Transcription of rRNA by RNA polymerase I Clusters of rRNA Genes • rRNA genes located at multiple sites along the DNA chromosomes • A single transcribed unit of DNA yields an initial RNA molecule of 45S in nucleolus, a special zone in nucleus • 45S RNA then processed to yield final

18S, 5.8S, and 28S subunits of rRNA

Myocilin Glaucoma • Myocilin (MYOC) = - multiple different mutations

1st glaucoma gene

The genetic code has a four code letters of DNA (A, T, G and C) in groups of 3 yield 4^3 (= 64) different combinations, enough to code for

20 amino acids

The polyubiquitinated proteins are recognized by a large, multi-subunit complex known as

26S proteasome

RNA polymerase I for class I genes (rDNA) to synthesize rRNA

28S rRNA & 18S rRNA

How many steps are in the elongation of prokaryotes?

Synonymous codons for Leucine: 5' - UUA - 3' 5' - UUG - 3' 5' - CUU* - 3' 5' - CUC* - 3' 5' - CUA* - 3' 5' - CUG - 3' There are six codons for leucine. Recognized by the same tRNA with anticodon

3'-GAI-5'

DNA template ("antisense" strand) is copied by RNA polymerase to synthesize complementary RNA: - RNA pol-catalyzed polymerization occurs by

3'-OH nucleophilic attack on alpha-phosphate of incoming rNTP

Prokaryotes - First base of the new mRNA transcript is not the first base of the open-reading frame (A of AUG codon) - First base of the new mRNA transcript begins the 5'-untranslated region (UTR) that comes before the ORF - Last base of the new mRNA transcript begins the ____ that comes after the ORF

3'-untranslated region (UTR)

Caspases (Cysteine Aspartases) - More than 10 members have been identified • Effector caspases:

3, 6, 7

(In prokaryotes) Streptomycin changes shape of

30S portion, causes code on mRNA to be read incorrectly.

Step 3 of initiation of prokaryotes: formation of initiation complex - (1) 50S subunit binds to

30S subunit to form the initiation complex

Step 1 of initiation of prokaryotes: binding of the mRNA to the 30S ribosomal subunit - (3) mRNA binds to ___ _____, guided by the Shine-Dalgarno Sequence

30S subunits

Wobble base pairing rules • When an amino acid is specified by several different codons, the codons that differ in either of the first two bases require different tRNAs • At least ____ _____ are required to translate all 61 codons (31 to encode the amino acids, 1 for initiation)

32 tRNAs

U1 binds the 5′ splice site and U2AF (U2 accessory factor protein) binds the _________.

3′ splice site

In prokaryotes the Shine-Dalgarno sequence consists of

4-9 purine nucleotides (purine-rich)

Retrotransposons make up most of the transposable elements in a human cell. In fact, these types of elements make up about _____ (or more) of the DNA in a genome

40%

Initiation in Eukaryotes Step 5: - The 60S subunit associates with the

40S subunit

Initiation in Eukaryotes Step 2: - (2) Forms a

43S pre-initiation complex

Eukaryotic Transcription of rRNA by RNA polymerase I Clusters of rRNA Genes • rRNA genes located at multiple sites along the DNA chromosomes • A single transcribed unit of DNA yields an initial RNA molecule of ________, a special zone in nucleus • 45S RNA then processed to yield final 18S, 5.8S, and 28S subunits of rRNA

45S in nucleolus

elF4F-mRNA complex binds to 43S pre-initiation complex to form

48S complex

What are important for splicing in eukaryotes?

5' Splice Site, 3' Splice Site, and Branch Site

Organization of promoters in prokaryotes:

5' to, or upstream of, gene coding regions

5' terminal portions of noncoding sequences are referred to as

5' untranslated regions (UTR)

DNA coding strand ("sense" strand) has the same sequence of the RNA & same

5'-> 3' direction

Prokaryotes - First base of the new mRNA transcript is not the first base of the open-reading frame (A of AUG codon) - First base of the new mRNA transcript begins the __________ that comes before the ORF

5'-untranslated region (UTR)

p53 - The most common genetic alterations in human tumors-mutated in more than ____ of all cancers

50%

(In prokaryotes) Erythromycin binds to

50S portion, prevents translocation-movement of ribosome along mRNA

(In prokaryotes) Chloramphenicol binds to

50s portion of ribosome and inhibits the formation of peptide bonds

A primary transcript (pre-mRNA) will not leave the nucleus until the final RNA processing is complete:

5′ cap, 3′ poly(A) tail, and removal of the introns (splicing)

Finally, splicing proceeds in three steps • First, the intron and exon are cut apart at the _______ and the free 5′ end of the intron loops around and is joined to the adenine at the branch site

5′ splice site

U1 snRNA recognizes the

5′ splice site at exon 1-intron boundary

The consensus recognition sequences for the 5′ splice site, 3′ splice site, and branch site are as shown above (residues in bold are most highly conserved as they are involved in the splicing mechanism)

5′ splice site: 5′-AG ↓ GUAAGU-3′ 3′ splice site: 5′-YYYYYYNC AG ↓ -3′ (Y=any pyrimidine; N=any nucleotide) branch site: 5′-U ACUA AC-3′

In eukaryotes, ribosomes bind the the __________ cap at the 5' terminus and scans downstream until they find the AUG initiation codon.

7-methylguanosine

In Burkitt's lymphoma, translocation of c-Myc from chromosome ______ places it close to Ig locus causing unregulated expression of c-Myc sufficient to drive cell proliferation.

8 to 14

Initiation of transcription in prokaryotes - Polymerase/DNA forms tight complex, melts DNA --> forms an open complex - Holoenzyme α2ββ'ωσ recognizes start site and first initiating nucleotide - Holoenzyme α2ββ'ωσ transcribes _________ before σ factor falls off and core enzyme (α2ββ'ω) - Continues elongation

8 to 9 bases

Caspases (Cysteine Aspartases) - More than 10 members have been identified • Initiator caspases:

8, 9, 10

Initiation of Eukaryotes - Hydrolysis of GTP is followed by release of eIFs and 60S subunit binds to the 40S subunit to from the

80S initiation complex

Chromosomal translocation - In chronic myelogenous leukemia (CML), Abl gene translocated from chromosome ___ fuses with Bcr gene at chromosome __ to form Philadelphia chromosome.

9;22

Mutation • Definition:

A change in the base sequence of DNA will produce change in the base sequence of mRNA and a change (may or may not) in the resulting protein.

Attachment (adsorption) of virion to the correct host cell - Before entering host cell, a virus generally must bind to a particular host receptor on the cell surface. - Most bacterial and plant viruses then abandon their protein coat; only their genetic material (DNA or RNA) enters the cell. - What is the Host Receptor?

A protein or a carbohydrate or a glycoprotein

Elongation Step 3 in prokaryotes: Translocation - The ribosome moves one codon toward the 3' end of the mRNA - Deacylated tRNA moves for the P to the E site and dipeptidyl-tRNA moves from the A to the P site. - The movement uses energy from GTP Hydrolysis. GTP is bound to EF-G or translocase. - ________ is vacant and is ready to accept next charged tRNA.

A site

Elongation: Step 1 in prokaryotes binding of incoming AA-tRNA • A tRNA carrying the next amino acid binds to the ____.

A site

Elongation: Step 1 in prokaryotes binding of incoming AA-tRNA • EF-Tu bound to GTP escorts the aminoacyl-tRNA to the ribosome. • GTP is hydrolyzed, the AA-tRNA is inserted into the ______. • EF-Tu-GDP complex leaves the ribosome. • EF-Tu does not interact with fmet-tRNAfmet and this initiator is never delivered to the A-site.

A site

Step 2 of initiation of prokaryotes: formation of pre-initiation complex - (2) All other incoming AA-tRNAs required for elongation bind to the

A site and subsequently to the P site.

Elongation in Eukaryotes - Step One: eEF1α-GTP escorts the tRNA carrying the 2nd amino acid to the A site - Step 2: GTP is hydrolyzed and eEF1α is released - Step 3: The peptidyl transferase activity of the large rRNA catalyzes peptide formation. - Step 4: GTP hydrolysis of eEF2-GTP facilitates translocation of ribosome one codon toward the 3' end of the mRNA. - Deacylated tRNA moves from the P to the E site and dipeptidyl-tRNA moves from the ____________. - A-site is vacant and is ready to accept next charged tRNA.

A to the P site

Apoptotic cells are efficiently phagocytosed by neighboring cells or macrophages. Which of the following DOES NOT normally happen in this process? A. The apoptotic cell releases some of its cytoplasmic content to induce a local inflammatory response. B. The apoptotic cell exposes phosphatidylserine at its surface, which interacts with receptor proteins on the surface of phagocytes via "bridging" proteins. C. The apoptotic cell loses or inactivates "don't eat me" signals. D. The apoptotic cell rounds up and detaches from its neighbors, which facilitates phagocytosis.

Cyclins modulate the progression of cells through the cell cycle by A. activating protein kinases that are critical regulators B. directly activating G proteins C. increasing the production of DNA polymerases D. phosphorylating histones E. Inducing synthesis of constitutively active forms of growth factor receptors

Formation of the ribosomal initiation complex for bacterial protein synthesis does not require: A) EF-Tu. B) formylmethionyl tRNAfMet. C) GTP. D) initiation factor 2 (IF-2). E) mRNA.

Glycophosphatidylinositol (or GPI) anchors are found A. at the carboxyl terminus of membrane proteins B. at the amino terminus of membrane proteins C. on the cytoplasmic side of membrane channel proteins D. on the amino terminus of the 28S of certain ribosomal complexes associated with secretion.

In eukaryotes which of the following amino acids is often hydroxylated after protein synthesis? A. Proline B. Tyrosine C. Arginine D. Methionine E. All of the above

In the absence of a trophic factor, the soluble pro-apoptotic protein Bad binds to the anti-apoptotic proteins Bcl-2 and Bcl-xl. What is the results of the binding of Bad to Bcl-2 nd Bcl-xl? A. Bax is able to form a channel in the outer mitochondrial membrane which results in the release of cytochrome c into the cytosol leading to apoptosis. B. The cell is prevented from going into apoptosis. C. It results in the down regulation of Bcl-2 expression. D. It prevents the activation of caspase-3. E. It prevents the activation of caspase-9.

Mutation of the codon GAU to which codon would MOST likely result in a protein with unaltered or minimally altered function? A. GAG B. GUG C. GCG D. GGG

Termination of polypeptide synthesis: A. Results from the release of a single release factor, eRF1, in eukaryotes. B. requires the binding of a complex of RF1 and RF2 to the A site in bacteria. C. Requires ATP hydrolysis D. requires binding of RF3 to the E site in bacteria.

The first stage of protein translation occurs when Met-tRNAi^Met base pairs with the mRNA being translated in the ribosomal: A. P site B. A site C. E site D. T site

Translocation of a gene can produce a fusion product with uncontrolled protein kinase activity. An example of this type of fusion protein is A. Tel/PDGFR B. Myc/Max C. Fos/Jun D. ErbB/Ras

When the ribosome reaches a stop codon on the mRNA, release factors act to change the specificity of peptidyl transferase so that the growing peptide chain is transferred to: A. Water B. elF4F C. Formyl-methionine D. UDP-GlcNAc E. The E site

Which of the following amino acids is sometimes carboxylated after protein synthesis in eukaryotes A. Glutamate B. Aspartate C. Phenylalanine D. Lysine E. Arginine

Which of the following is proapoptotic? A. Bax B. Bcl-2 C. Rb D. Akt E. Ras

Which of the following is required for the initiation of protein synthesis in the cytosol of eukaryotic cells? A. A 40S ribosomal subunit B. Initiation Factor (IF)-2 C. Methionyl-tRNAf^Met D. Uracil Triphosphate (UTP) E. Elongation Factor (EF)-2

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA • CPSF (cleavage and polyadenylation specificity factor) binds the

AAUAAA site

Ubiquitin-proteasome pathway • Ubiquitin is released and reused. • Requires _____ hydrolysis

ATP

____ hydrolysis is required for the release of the unfolded protein from chaperones as well as for folding within the chaperonin.

ATP

Initiation of Eukaryotes - The 40S subunit in association with bound tRNA and eIFs scan mRNA to identify initiation AUG codon. ____ _____ provides energy.

ATP hydrolysis

Major Mechanism for RNA Export Through the NPC •The export mechanism for RNAs is different • Ran-GTP independent • Instead, _____ dependent • Association of mRNA with large export complex • Quality control of processed mRNA • RNA helicase on cytoplasmic face of NPC releases mRNA from export complex

ATPase

Initiation in Eukaryotes Step 3: The elF4F complex binds to mRNA - eIF4F complex: - elF4A is an

ATPase and a helicase

Chromosomal translocation - _____ is a tyrosine kinase

Abl

Frameshift Mutation: "Read through" of termination codon due to a frameshift mutation results in the production of abnormally long α-globin chains, e.g.,

Abnormal Hemoglobin Wayne

Ubiquitin-proteasome pathway Step 2:

Activated ubiquitin is transferred to ubiquitin conjugating enzyme E2.

Cdc25C phosphatase

Activates vertebrate mitotic CDKs

M-CDK is regulated by phosphorylation and dephosphorylation. Thr-161:

Activating phosphorylation

Prokaryotes: Regulation is simple and rare (e.g., Lac operon) and controlled only by ____ ______ (e.g., Crp ± cAMP; LacI ± allo-lactose)

Activators / Repressors

Altered Wnt Signaling in Glaucoma • Wnt inhibitors in glaucoma - Increased SFRP1 expression - Increased DKK1 expression • Blocked Wnt signaling in trabecular meshwork •Target validation - ____ or _____ elevates IOP •New glaucoma model

Ad5.SFRP1 or Ad5.DKK1

Decoding mRNA by transfer RNA (tRNA) - _______ that translate genetic information from mRNA into proteins

Adaptors

Amino acid activation: Step Two

After charging, the anticodon in the tRNA base pairs with codon in the mRNA specifying the amino acid attached to the tRNA. This ensures proper placement of the amino acid in a growing polypeptide.

Retrovirus replication: - Step 2:

After dsDNA retrovirus genome copy is made by RT, the dsDNA integrates into the host chromosome by actions of a virus-specific integrase enzyme. The dsDNA is flanked by two long terminal repeats (LTR) that facilitate the insertion.

Degenerate: An amino acid may be specified by more than one codon. An example is

Ala is coded by GCU, GCC, GCA, GCG

Gene Therapy - DNA Targets •Introns

Alternative splicing

• The synthesis of a polypeptide does not produce a functional protein. • A polypeptide must be folded into its native conformation. • The newly synthesized proteins have to be delivered to appropriate destinations. • Proteins undergo certain modifications to attain their biologically active forms. • Abnormal, damaged, unwanted as well as regulatory proteins are degraded. • Functions:

Alters protein stability, activity, localization, interaction with other biomolecules, signal transduction, metabolism, cell division, cell death

What is an example of retrotransposons?

Alu elements

Formation of amyloid aggregates is associated with a variety of diseases, such as ______ disease, ______ disease and _____ _ _______.

Alzheimer's, Parkinson's, Type 2 diabetes

Amino acid activation • The aminoacyl-tRNA synthetases esterify 20 amino acids to their corresponding tRNAs. • These synthetases must be specific for both amino acid and tRNA.

Amino acid + ATP + tRNA → Aminoacyl-tRNA + AMP + 2Pi

Properties of the genetic code - Collinear

Amino acid sequence of a protein is defined by a linear sequence of contiguous triplet codons

What is the aggregating protein in Type 2 diabetes?

Amylin

What is the aggregating protein in Alzheimer's Disease?

Amyloid-β

Properties of the genetic code - Degenerate:

An amino acid may be specified by more than one codon

Amino acid activation: Step One

An aminoacyl-tRNA synthetase couples an amino acid to the terminal Adenosine in the tRNA that has proper anticodon for the amino acid. The high energy ester bond is required for subsequent peptide bond formation.

_____apoptotic Bcl-2 family proteins maintain mitochondrial integrity.

Anti

Vertebrates - Adapter:

Apaf-1 (apoptosis promoting activator 1)

M cyclins:

Appear at the beginning of G2 and accumulate through G2

What is the fifth step in the life cycle of a typical virus?

Assembly of new virions

(-)ssRNA animal viruses: Step 1: Synthesis of (+) ssRNA Step 2: (+) ssRNA is used to make more (-) ssRNA. (-) ssRNA is used to make (+) ssRNA ("mRNA") --> translated to proteins Step 3 ?

Assembly of nucleocapsids

What is the first step in the life cycle of a typical virus?

Attachment (adsorption) of virion to the correct host cell

What are the steps of viral replication?

Attachment → penetration → uncoating → early genes → genome replication → late genes → assembly → release of virions

Hallmarks of cancer - Limitless replicative potential:

Avoid replicative senescence and have unrestricted proliferative capacity

Bcl-2 was first identified in

B-cell lymphoma

Cellular Protein Synthesis Proceeds in which direction? A. carboxyl to amino terminus B. Amino to carboxyl terminus C. 3' to 5' D. 5' to 3'

Cellular changes that are involved in cancer progression include all of the following EXCEPT: A. resistance to cell death mechanisms. B. induction of apoptosis C. evasion of growth suppression. D. facilitation of invasion and metastasis E. All of these are involved

Growth factors are important for the regulation of cell cycle. If growth factors are removed prior to restriction point the cell cycle will A. be independent of cyclin D B. cease at G1 and enter G0 C. continue to proceed to S D. revert back to mitosis E. stop in G2

In proteasomes, ubiquitin A. is degraded B. is released and recycled C. is phosphorylated D. has a GTP group added

N-glycosylation of proteins A. begins in the Golgi complex and continues as the proteins are trafficked to the ER. B. occurs initially by transfer of a core oligosaccharide structure from dolichol phosphate to an asparagine side chain of the protein. C. Only occurs on proteins destined for secretion from the cell. D. Occurs primarily in the cytosol.

The correctly folded three-dimensional configuration of a protein is determined primarily by the A. sequence of nucleotides of its gene B. primary sequence of its amino acids C. chaperones with which it interacts D. pathway by which if folds

The enzyme protein disulfide isomerase, which facilitates breakage and reformation of disulfide bonds, is located primarily in the A. cytosol B. lumen of the endoplasmic reticulum C. matrix of the mitochondria D. lumen of lysosomes

The progression from metaphase to anaphase is triggered by A. activation of protein kinases by M-CDK B. ubiquitin-mediated proteolysis of key proteins C. binding of an inhibitory protein to M-CDK D. depolymerization of the mitotic spindle.

The retinoblastoma protein binds to and inhibits A. oncogenes B. transcription factor E2F C. cyclin-dependent kinase D. p53 E. p21

What is the structural signal that targets proteins to lysosomes? A. ubiquitin B. mannose-6-phosphate residues in N-linked oligosaccharides C. a signal sequence of 10-15 hydrophobic amino acids D. one or more positively charged residues, usually near the amino terminus, preceding the hydrophobic sequence

Which of the following caspases is an effector caspase involved in the execution of apoptosis? A. caspase-1 B. caspase-3 C. caspase-4 D. caspase-8 E. caspase-9

Which of the following inhibits cyclin A/Cdk2 activity? A. Rb B. p21 C. p53 D. INK4

Which of the following processes is indicative of cells undergoing apoptosis? A. Cell swelling B. Cell shrinkage and chromatin condensation C. Inflammatory response D. Plasma membrane lysis E. Random cleavage of DNA

With the messenger RNA (mRNA) codon would the transfer RNA (tRNA) 5'-UAC-3' be able to form a codon-anticodon base pairing interaction? A. 5'-AUG-3' B. 5'-GUA-3' C. 5'-UAC-3' D. 5'-CAU-3' E. 5'-GAU-3'

A 50-year-old man was diagnosed with follicular lymphoma. The karyotypic analysis revealed t(14:18) chromosomal translocation which caused increased production of a gene product that inhibits apoptosis. The affected gene is:

Bcl-2

Mitochondrial or intrinsic cell death pathway - The _____ family proteins regulate mitochondrial integrity and cytochrome c release

Bcl-2

Vertebrates - Regulator:

Bcl-2 (inhibits cell death)

Regulation of mitochondrial integrity by

Bcl-2 family proteins

• There are more than 20 members in the Bcl-2 family - Anti-apoptotic Bcl-2 family proteins:

Bcl-2, Bcl-xl, Mcl-1

Imatinib mesylate or Gleevec inhibits

Bcr-Abl tyrosine kinase activity

Gleevec (STI-571) blocks proliferation of chronic myeloid leukemia cells by inhibiting the following protein kinase ________.

Bcr/Abl

Retrotransposons and disease - Over-representation of diseases caused by the X chromosome

Because the X chromosome has many LINE and SINE elements. Some cases of de novo hemophelia are a result of retrotransposons

Activation of caspase-8 by the extrinsic pathway causes cleavage of

Bid

Eukaryotic - elF4G

Binds to eIF4E and to poly(A) binding protein (PABP); part of the eIF4F complex

Eukaryotic - elF4E

Binds to the 5' cap of mRNA; part of the eIF4F complex

Eukaryotic - elF1

Binds to the E site of the 40S subunit

Eukaryotic gene control region (for mRNA coding genes):

Bipartite promoter + Regulatory DNA sequences

Eukaryotic RNA Polymerase III transcribes genes from a Class III gene promoter - TFIIIC = two subunits τA and τB, binds to A and B blocks. - This induces TFIIIB complex (______) to bind promoter near the start site

Brf1, Bdp1, TBP

Glycophosphatidylinositol (GPI) anchors: Proteins can be anchored to the outer face of the plasma membrane by glycolipids attached to their

C-terminus

A 4-year-old girl Annie was diagnosed with β-thalassemia. Genetic analysis revealed that codon 17 of the β-globin gene has been changed from UGG to UGA. What type of mutation was detected in Annie's β-globin gene? A. Silent Mutation B. Missense Mutation C. Nonsense Mutation D. Frameshift Mutation

An early step in the post-translational processing of secreted proteins in eukaryotes is A. proteolytic removal of the first two or three amino acid residues from the amino terminus by signal recognition particle. B. attachment of a mannose-rich oligopeptide to asparagine side chains. C. proteolytic cleavage of the signal sequence by signal peptidase. D. attachment of mannose-6-phosphate to the protein E. Formation of disulfide bonds

Compared to their normal counterparts, leukemic cells A. continue to differentiate B. fail to proliferate C. fail to undergo apoptosis D. induce widespread apoptosis

Cycloheximide causes a significant disruption of which of the following process of eukaryotic A. Blocks the initiation of protein synthesis. B. Causes premature chain termination. C. Inhibits peptidyl transferase activity of 60S ribosome. D. Inhibits release of polypeptide. E. Inhibits binding of aminoacyl transfer RNA.

In the synthesis of a charged tRNA A. There is only one aminoacyl-tRNA synthetase for all tRNAs. B. Each amino acid is added via an ester linkage to the 5' end of the tRNA. C. There is a separate aminoacyl-tRNA synthetase for every amino acid. D. The selection of the amino acid to be added is based exclusively on the anticodon sequence of the tRNA. E. ADP and Pi are included in the products formed.

Proteins that facilitate the folding of other proteins are called A. Foldases B. Binding proteins C. Chaperones D. Escorts

Separase initiates sister chromatid segregation at anaphase by cleaving A. APC B. Cdc20 C. Cohesin D. Cyclin B E. Securin

The expression of the following anti-apoptotic protein is elevated in certain tumors. A. p53 B. Bax C. Bcl-2 D. Cytochrome C E. Rb

The proteins shaped like a "double chamber" that are involved in protein folding are called A. Hsp70 proteins B. protein disulfide isomerases C. chaperonin proteins D. peptidyl prolyl isomerases

What is the function of the Golgi Apparatus? A. Storage and replication of DNA B. Hydrolytic degradation of biomolecules C. Post-translational modification of secretory and membrane proteins D. Source of anti-oxidation molecules such as catalase E. Production of ATP

What is the primary substrate for caspase-9 during the process of initiating apoptosis? A. caspase-1 B. Apaf-1 C. Pro-caspase-3 D. cytochrome c E. Bax

Which of the following events in usually associated with necrosis? A. Nuclear lamin proteolysis B. Membrane blebbing C. Rapid lysis of the cell membrane followed by an inflammatory response. D. Fragmentation of DNA by nucleases. E. Engulfment of the apoptotic bodies by macrophages.

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA • CF endonuclease cuts growing RNA polymer 10~30 bases downstream of AAUAAA, cutting just after the

CA dinucleotide

cAMP bind to Crp leads to DNA binding ("allostery") - Crp (cyclic AMP receptor protein) is also called __________ - Crp/CAP = "global control protein" affecting multiple sugar-related operons

CAP (catabolite activator protein)

Human T-Cell Leukemia Virus (HTLV-1) - Endemic in Japan, South America, and Africa - Affects _______ (like HIV ---> Gallo called it "HTLV-III" in 1980s)

CD4+ T-cells

CDC2 is similar to

CDK1

C. elegans - Effector:

CED-3 (causes cell death)

C. elegans - Adapter:

CED-4 (facilitates cell death)

C. elegans - Regulator:

CED-9 (inhibits cell death)

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA - CPSF & CST provide platform for assembly of

CF (cleavage factor, not shown), poly(A) polymerase, and PABP (poly(A)-binding protein).

Cystic fibrosis is caused by rapid degradation of _____.

CFTR

Defects in mRNA Transport Cause Diseases - The most common form of DM in adults, DM type I, is an autosomal dominant disease caused by an abnormal ___ ______ in the 3′ untranslated region (UTR) of the DM protein kinase mRNA (DMPKCUG+) - Toxic ____ repeats within DMPKCUG+ result in nuclear RNAs that are resistant to export

CUG expansion

Retrotransposons and Disease - Because some are still active in the genome they can cause disease • Insert in a gene - ????

Can cause disruption of a gene function

What is the replication cycle of (-) ssRNA genome?

Cannot be used directly to synthesize viral proteins; Therefore, the virus carries the RNA-dependent RNA polymerase inside virion!

miRNAs in Disease • Complex regulatory circuits of miRNAs • Dysregulation of miRNA expression in variety of diseases • Viral pathogenesis • ______ hypertrophy

Cardiac

Human T-Cell Leukemia Virus (HTLV-1)

Carries no oncogene nor uses insertional mutagenesis

Gene Therapy - Genome Editing • CRISPR/____ genome editing

Cas

Vertebrates - Effector:

Casp9 and Casp3 (3 - promotes cell death)

Initiator caspases contain a long pro-domain • Caspase recruitment domain (CARD):

Caspase-9

CAD:

Caspase-activated DNase

The process of apoptosis is broadly divided into two phases: Initiation phase: ??? Execution phase:

Caspases become active

The process of apoptosis is broadly divided into two phases: Initiation phase: Execution phase: ???

Caspases trigger degradation of critical cellular components

Chromosome Segregation - Phosphorylation of APC facilitates its binding to

Cdc20

Chromosome Segregation - ______ binds to anaphase promoting complex (APC) and activates the proteasomal activity of APC.

Cdc20

Chk1 and Chk2 phosphorylate and inactivate

Cdc25

Cip1

Cdk2 interacting protein 1

Growth factors ---> (Ras/Raf/MEK/ERK pathway) ---> synthesis of D-type cyclins --->

Cdk4, 6/ Cyc D

p53 absent or mutated:

Cells accumulate DNA damage, can cause mutations, and progress to malignant tumor.

Point mutation

Change in a single base pair in DNA

Characteristics of apoptosis - activation of endonuclease:

Cleavage of genomic DNA into multiples of 180 bp internucleosomal fragments

Gene Therapy - Genome Editing • Being explored for large number of diseases • Clinical trials and approved therapies What does CASPER stand for?

Clustered Regularly Interspaced Short Palindromic Repeats

Ubiquitin-proteasome pathway - Covalently linked to proteins targeted for degradation via an _____ _____ pathway.

CovalentlyATP-dependent

CDK4 & CDK6- which cyclins?

Cyclin D

CDK2 - which cyclins?

Cyclin E / Cyclin A

When released from Rb, E2F transcribes Cyclin E and Cyclin A causing activation of _______ and _____.

Cyclin E-CDK2 (G1/S CDK) ; Cyclin A-CDK2 (S-CDK).

An apoptosome is a complex containing multiple subunits of A. Apaf-1 B. Apaf-1 and caspase-9 C. Apaf-1 and caspase-3 D. Apaf-1, caspase-9, and cytochrome c.

Charging of a tRNA molecule with the correct amino acid is dependent on A. the anticodon loop B. the variable loop C. the codon of the mRNA D. an aminoacyl synthetase specific for that tRNA

Chronic myeloid leukemia is caused by a translocation that creates: A. A proto-oncogene B. A fusion protein that acts like a transcription factor C. A protein that increases growth factor production D. A fusion protein that deregulates the cell cycle of myeloid white blood cells E. A deletion of a whole chromosome

In response to many cell death stimuli, the molecule that is released from the mitochondria and binds to Apaf-1 is A. ATP synthase B. citric acid C. ubiquinone D. cytochrome C E. Bid

Most of the cyclins are induced and degraded periodically during the cell cycle. One cyclin controlled by growth factor is A. cyclin A B. cyclin B C. cyclin C D. cyclin D E. cyclin E

Peptide bond formation is translation occurs by A. an enzyme catalyzed reaction B. a tRNA catalyzed reaction C. a tRNA synthase catalyzed reaction D. an rRNA catalyzed reaction

The coding sequence (CS)-1 is the normal coding strand of the gene. A mutation has occurred in CS-1 resulting in CS-2. The reading frame begins with the first nucleotides shown in the sequences below. What is the consequence of the mutation shown by CS-2? CS-1: 5'- GATTCATGTTCGGACTGGTGTAT-3' CS-2: 5'-GATTCATGTTCGGACTGGAT-3' A. A single amino acid is added in the coding sequence 2. B. A single amino acid is replaced by another amino acid. C. This is nonsense mutation that results in premature chain termination. D. A single amino acid is missing in the coding sequence 2. E. Splicing of the transcript is abnormal.

The factor that escorts the aminoacyl tRNA to the eukaryotic ribosome and then releases it with GTP hydrolysis following the correct codon-anticodon base pairing is A. elF1 B. eRF1 C. elF2 D. eEF1α

The mRNA sequence 5'-GCG ACG UCC-3', is being translated by a ribosome. Which of the following sequences represent the anticodons of the aminoacyl-tRNAs that were used (in order) during translation? A. 5'-UCC-3' 5'-ACG-3' 5'-GCG-3' B. 5'-GGA-3' 5'-CGU-3' 5'-UCC-3' C. 5'-GCG-3' 5'-ACG-3' 5'-UCC-3' D. 5'-CGC-3' 5'-CGU-3' 5'-GGA-3' E. 5'-CGC-3' 5'-GCA-3' 5'-UCC-3'

The phase of cell cycle that corresponds to the interval between mitosis and initiation of DNA replication is referred to as A. M phase B. S phase C. G0 phase D. G1 phase E. G2 phase

The phenylalaine (Phe) residue of Phe-tRNA^Phe was chemically converted to Tyrosine (Tyr). When Tyr-tRNA^Phe was mixed with components of the protein synthesis machinery where will the Tyr from Tyr-tRNA^Phe be incorporated during the protein synthesis? A. It cannot be incorporated anywhere in the protein. B. It will be incorporated where Tyr normally occurs. C. It will be incorporated randomly where either Tyr or Phe occurs. D. It will be incorporated where Phe normally occurs. E. It will cause premature termination of the protein.

What is the biochemical advantage when a cell uses polysomes? A. Many fewer translation errors B. Far less energy expended C. Fewer molecules of ribosomal RNA needed D. Much faster protein synthesis E. Containment of all of the newly synthesized protein in an organelle

When p53 increases in response to DNA damage, which of the following events occurs? A. p53 induces transcription of Cdk4 B. p53 induces transcription of cyclin D C. p53 binds E2F to activate transcription D. p53 induces transcription of p21 E. p53 phosphorylates jun.

Which antibiotic or toxin mechanism is INCORRECT? A. Tetracyclines block the A site of the ribosome. B. Cycloheximide blocks the peptidyl transferase activity of eukaryotic ribosomes. C. Chloramphenicol blocks peptidyl transfer in bacteria. D. Puromycin blocks the P site of bacterial ribosomes.

Which of the following features is common between the prokaryotic and eukaryotic ribosomes in translation initiation? A. They both use an initiator tRNA that carries formylmethionine. B. They both bind to the 5' end of the mRNA and move forward to find the start codon. C. They both recognize the start codon by interacting with the Shine-Dalgarno Sequence. D. They both interact with various translation initiation factors. E. All of the above.

Which of the following features is different between the prokaryotic and eukaryotic ribosomes in translation initiation? A. GTP hydrolysis is required for bacterial initiation but not for eukaryotic initiation. B. Many more initiation factors are required for bacteria than for eukaryotes. C. In bacteria, the large subunit of the ribosome binds after the mRNA; in eukaryotes, the large subunit binds before the mRNA. D. In bacteria, base-pairing between the mRNA and a rRNA determine which start codon to use; in eukaryotes, the small subunit begins scanning the mRNA at the 5' cap until it reaches the first start codon.

Which of the following groups anchors proteins to the outer surface of the plasma membrane? A. Myristate B. Farnesyl C. Palmitate D. Glycolipid

Which of the following is an essential characteristic of the intrinsic pathway of apoptosis? A. Activation of caspase 8 B. Cleavage of the proapopototic factor, BID by caspase 8 C. Involvement of Fas-associated death domain protein (FADD) D. Release of cytochrome c from mitochondria E. Death receptor pathway

Which of the following is not a feature of the genetic code? A. Triplet B. Degenerate C. Non-overlapping D. Ambiguous

Which of the following is not an example of post-translational modification? A. Ubiquitination B. N-Myristolyation C. Glycosylation D. Polyadenylation

Which of the following is seen when a single mRNA is simultaneously translated to form multiple copies of a polypeptide chain? A. Phagosome B. Multisome C. Nucleosome D. Polysome

Which one of the following is a substrate of ATR? A. Cdc25A B. Cdc25C C. Cyclin B D. Chk1 E. Rb

DD and death effector domain (DED) and interact with pro-caspase-8 via DED

Activation of initiator pro-caspase-8 - Binding of death ligands to the receptor triggers receptor oligomerization. - Death domain (DD) of the receptors recruits other DD-containing adaptors (e.g., FADD, TRADD). - The adaptors contain both DD and death effector domain (DED) and interact with pro-caspase-8 via DED - Binding of initiator pro-caspase-8 to ____ causes autoproteolytic activation of initiator caspase-8.

DISC

Genome Replication: Retrovirus have a _____ intermediate created by viral RNA-dependent DNA polymerase or "RT", integrate into host genome.

DNA

Hepatitis B virus (HBV) - Type: ____ - Family: Polyomarvirus - Cancers caused: Merkel cell carcinoma

DNA

Human Papilloma Virus (HPV) - Type: ____ - Family: Papillomavirus - Cancers caused: Cervial, other epithelial

DNA

Introns are only present in

DNA & pre-mRNA

p53 is activated in response to

DNA damage

ATM (ataxia telangiectasia mutated), ATR (AT- and Rad3-related) and p53 play important roles in

DNA damage checkpoints

Retroviruses - Unlike other (+) ssRNA viruses, retroviral replication uses a

DNA intermediate

DNA replication checkpoint prevents initiation of mitosis until

DNA replication is complete

Promoter =

DNA sequence located 5' to gene's coding sequence where the RNA polymerase binds to initiate transcription

S phase (6-8 hours)

DNA synthesis doubles the amount of DNA in the cell. RNA and protein also synthesized.

Exons are present in

DNA, pre-mRNA, and mRNA

Leucine Zipper

DNA-binding motifis an extended amphipathic alpha-helical polypeptide with hydrophobic amino acids concentrated to one side (e.g., Leu every 7th position)

• One example of a specific splicing variant associated with cancers is in one of the human DNMT (DNA methyl transferase) genes • Three DNMT genes encode enzymes that add methyl groups to DNA, a modification that often has epigenetic regulatory effects • Several abnormally spliced _____ mRNAs are found in tumors and cancer cell lines • Expression of two of these abnormally spliced mRNAs in mammalian cells caused changes in the DNA methylation patterns in those cells • Cells with one of the abnormal mRNAs also grew twice as fast as control cells, indicating a direct contribution to tumor development by this product

DNMT3B

Role of miRNA in Limb Development - Conditional _____ knockout showing role of Gd5 in mouse embryonic limb development (E13)

Dicer

MicroRNA Processing • Nuclear export to cytosol (via exportin-5) • Processed by ___ _____ to form miRNA

Dicer complex

The Identification of Gain-of-Function and Loss-of-Function Cancer Mutations Has Traditionally Required

Different Methods

Organization of prokaryotic promoters -35 box:

Different, highly conserved 6 base sequence on DNA coding strand. These two conserved sequences are recognized by DNA-dependent RNA polymerase with sigma (s) factor.

MicroRNA Processing • Primary microRNA (pri-miRNA) with hairpin loop • Processed by _____ ______ to pre-miRNA with hairpin loop

Drosha complex

The kinase that phosphorylates lamin is activated by A. p53 B. cyclin D C. ras D. ERK E. cyclin B

What reaction mechanism provides a role in bacterial cell proofreading during elongation, ensuring the correct aminoacyl-tRNA is added to the A site? A. Phosphorylation from ATP B. Peptide bond formation C. Formation of a temporary disulfide bond D. Adenylyation E. GTP Hydrolysis

Which one of the following is a target of cyclin D-Cdk4/6 complex? A. E2F B. p21^Cip1 C. p53 D. Cyclin E-CDK2 E. Rb

Human Papilloma Virus (HPV) - Host cell growth maintained by viral proteins ____ and _____

E6 and E7

Proto-oncogenes serve important cellular functions - Growth factor receptors:

EGFR (ErbB), PDGFR

Alternative Splicing of Fibronectin mRNA • This 75 kb gene contains multiple exons; splicing of fibronectin varies by cell type • The ____ and ____ exons (green) encode binding domains for specific proteins on the surface of fibroblasts. The fibronectin mRNA produced in fibroblasts often includes the _____ and ______ exons (cellular FN), whereas these exons are spliced out of mRNA in hepatocytes (soluble FN)

EIIIB;EIIIA

Replication of dsDNA Viruses (e.g., HSV) - Synthesis of late proteins

Early Proteins + cell enzymes ---> Replication of virus DNA --> Progeny virus --> transcription of late genes (fraction of the viral genome transcribed after initiation of viral DNA synthesis) --> late mRNAs --> translation --> late proteins --> assembly of nucleocapsids

What is the second step in the life cycle of a typical virus?

Entry (penetration) of the virion or viral genome

Hereceptin is a monoclonal antibody against the oncogene protein

ErbB-2

Extrinsic or receptor-initiated apoptosis - Death domain (DD) of the receptors recruits other DD-containing adaptors (e.g., _____, ____). - The adaptor can interact with initiator caspase-8.

FADD;TRADD

Bacterial - IF2

Facilitates binding of fMet-tRNA^fMet to 30S ribosomal subunit

Cell cycle arrest at the DNA damage checkpoint - Cells are arrested at the

G1, S, or G2 phase

Passage through the restriction point - In response to growth factors, D type cyclins are synthesized and activate _______ which phosphorylates Rb.

G1/CDK

p53 induces p21, a CKI which inhibits _____ and _____.

G1/S-CDK ; S-CDK

Common upstream elements include

GC box, CAAT box, AP1 element, and Octamer element.

Glucocorticoid-Induced Glaucoma • One side effect of GC therapy is development of ______ (GC-OHT) and iatrogenic glaucoma • GCs damage the trabecular meshwork (TM) causing increased IOP • Individual differences in susceptibilities to develop GC-OHT - 30-40% of normal population can develop GC-OHT - Almost all glaucoma patients can develop GC-OHT • Susceptibility to GC-OHT due to differential GR mRNA splicing?

GC-induced ocular hypertension

Altered Wnt Signaling in Glaucoma - Discovery of new glaucoma therapy - _____ inhibitors

GSK3b

pre-mRNA Processed by Splicing (Removal) of Introns - There are several classes of introns - The most frequent class of intron in eukaryotic nuclear genes is the ______ (or _____ in RNA code) group of introns.

GT-AG;GU-AG

Elongation Step 3 in prokaryotes: Translocation - The ribosome moves one codon toward the 3' end of the mRNA - Deacylated tRNA moves for the P to the E site and dipeptidyl-tRNA moves from the A to the P site. - The movement uses energy from _____ _____. _____ is bound to EF-G or translocase.

GTP hydrolysis

In the elongation of prokaryotes, step one is the binding of an incoming AA-tRNA. What is the rate limiting step?

GTP hydrolysis

Elongation in Eukaryotes - Step One: eEF1α-GTP escorts the tRNA carrying the 2nd amino acid to the A site - Step 2:

GTP is hydrolyzed and eEF1α is released

Initiation in Eukaryotes Step 2: - (1) The initiator tRNA^Met complexed with _______ along with _______ bind to the 40S ribosomal subunit.

GTP-elF2;elF5B-GTP

Eukaryotic - elF2

GTPase; facilitates binding of initiating Met-tRNAMet to 40S ribosomal subunit

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA - CST (cleavage stimulation factor) binds the

GU-rich tract

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA • Genes destined to create polyadenylated mRNA usually have 5'-AATAAA-3' site in coding strand, faithfully copied by RNA pol II into "tail signal"5'-AAUAAA-3' close to 3'-end • RNA poly II continues past tail signal to also create CA dinucleotide &

GU-rich tract

Eukaryotes • The vast majority of introns initiate with ____ and terminate with _____.

GU; AG

HPV vaccine

Gardasil

Environment? + Glucose , - Lactose

Glucose is present so you do not make cAMP and do not get CRP binding. No lactose so the repressor is bound and is blocked. So the signal is off.

Acetylation of Histone Tails Disaggregates Nucleosomes: • Closely-packed nucleosomes are stabilized by binding of histone tails to histones in the next nucleosome. • When _______ is acetylated by histone acetyl transferase (HAT), it no longer binds to adjacent histones. This promotes disaggregation of neighboring nucleosomes. (Histone H1 binds to linker DNA between nucleosomes but is not shown in this figure for sake of clarity.)

H4 tail

What are the homeodomain proteins - "master regulators" in development??

Helix-turn-helix

The life cycle of a virus 5. Assembly of new virions 6. Release of new virions - Capsid components self-assemble and associate wxith nucleic acid to form nucleocapsid • Non-enveloped viruses?

Host cell bursts and new virions released

Alternate Splicing Leads to

Human Genetic Diseases

What is the aggregating protein in Huntington's disease?

Huntingtin

Conversion of proto-oncogenes to oncogenes - Point mutation:

Hyperactive or constitutively active protein

Step 2 of initiation of prokaryotes: formation of pre-initiation complex - (1) 30S ribosomal complex binds _______________ forming "pre-initiation complex."

IF-2 bound to GTP and fmet-tRNA^fmet

p53 normal:

In response to DNA damage, p53 prevents tumor formation by causing cell cycle arrest to allow time for DNA repair. If repair fails, in can induce apoptosis to prevent accumulation of cells with damaged DNA.

ICAD

Inhibitor of Caspase Activated DNAse

M-CDK is regulated by phosphorylation and dephosphorylation. - Thr-14 and Tyr-15:

Inhibitory phosphorylation

Explain the 4 stages of RNA transcription in prokaryotes: - Stage 2

Initiation of transcription

If a tRNA contains _______ (I), it can H-bond with U, C and A.

Inosinate

Promoter insertion:

Integrate in promoter of proto-oncogene

Hallmarks of cancer - Ability to invade and metastasize:

Invasion and metastasis are the hallmarks of malignant tumors and the root cause of morbidity and mortality

Proteins that usually reside in the ER posses _______ sequence at the C-terminus.

KDEL (Lys-Asp-Glu-Leu)

What is the most important group of retrotransposons in human?

LINEs

What are the viral proteins in Epstein Barr Virus (EBV)?

LMP-1 & EBNA-2 involved in transformation

Enhancers enhance rate of transcription at a distance:

Looping model

_____ is a marketed drug that helps patients with loss of PRE65.

Luxturna

Methylation of ___ and ____ alters gene expression, DNA repair, protein degradation

Lys;Arg

_____ or ______ phosphorylates and activates condensins.

M-Cdk or Cyclin B/Cdk1

What is an example of gene amplification?

Myc, ErbB2, Mdm2, cyclin D

Cytogenetic analysis of the tumor cells from a patient show an integrated homogeneously staining region (HSR) on chromosome 4. Which of the following oncogenes is most likely involved in this case?

N-MYC

______ is amplified in neuroblastoma.

N-Myc

What are the classifications of glycosylation?

N-linked or O-linked

Protein targeting to mitochondria - The information for targeting protein from the cytosol to the mitochondria is contained in the targeting sequence at the

N-terminus

G2 Phase (3-4 h)

No DNA synthesis. RNA and protein synthesis continue.

Environment? - Glucose , - Lactose

No glucose is present so we signal cAMP but it is blocked b/c no lactose. So signal is off.

Eukaryotic DNA packaged into chromatin (creates unique level of control of masking or unmasking genes for transcription) versus Prokaryotes:

No packaging of DNA into histones or chromatin

Retrovirus replication: - Step 3:

Once integrated into the host chromosome, retroviral DNA is transcribed as any other gene in the host cell by host cell RNA polymerase. The retrovirus RNA is normally processed by addition of a 7-methyl-G-cap and a poly(A) tail.

Tumor viruses can cause cancer: - Viruses capable of initiating cancer are called "_______"

Oncoviruses

Protein targeting to mitochondria -When does Mitochondrial targeting start?

Only after a precursor protein is completely synthesized and released from the ribosome.

Non-Hereditary Retinoblastoma - occasional cell inactivates one of its two good Rb genes - occasional cell inactivates its only good Rb gene copy --> excessive cell proliferation leading to retinoblastoma Result: ???

Only bout 1 in 30,000 normal people develop one tumor in one eye

Reading frame - A new codon begins every three nucleotide residues. - The first codon establishes the reading frame. - Any mRNA has three possible reading frames - _____ _____ _____ contains a series of triplets without any termination codon.

Open reading frame

Elongation Step 2 in prokaryotes: peptide bond formation - The "uncharged" tRNAfmet is now bound to the ____ and the growing peptide is now located at the A-site.

P site

Initiation in Eukaryotes Step 5: - The 80S initiation complex is formed with the tRNAiMet base paired with the AUG initiation codon at the ribosomal

P site

Termination in Eukaryotes - Translation is terminated when termination codons (UAA, UAG, or UGA) are at the A site. - The release factor eRF3 bound to GTP escorts eRF1 to the A site. i. eRF3-GTP is hydrolyzed, and the peptide bond is cleaved from the tRNA in the _____.

P site

Step 1 of initiation of prokaryotes: binding of the mRNA to the 30S ribosomal subunit - (4) AUG is positioned in the

P site, the binding site for fmet-tRNA^fmet

Elongation Step 3 in prokaryotes: Translocation - The ribosome moves one codon toward the 3' end of the mRNA - Deacylated tRNA moves for the ____ to the ____ and dipeptidyl-tRNA moves from the A to the P site.

P;E site

Replication of dsDNA Viruses (e.g., HSV) - Synthesis of early proteins

Parental DNA ---> Transcription of earrly genes (fraction of the viral genome transcribed prior to initiation of viral DNA synthesis) ---> early mRNAs --> Translation ---> early proteins

Gene Therapy Future - ________ _________ - prevention: early detection of patients at risk, improve preventive measures (individual/collective) - diagnosis: accurate disease diagnosis enabling individualized treatment strategy - treatment: improved outcomes through targeted treatment and reduced side effects

Personalized medicine

What is the most common modification of individual amino acids?

Phosphorylation / Dephosphorylation

Mitotic protein kinases - Fragmentation Golgi Apparatus:

Phosphorylation of Golgi matrix proteins

Mitotic protein kinases - Spindle formation:

Phosphorylation of centrosome, kinetochore, and microtubule-associated proteins

Mature eukaryotic mRNA has 5' cap structure and ______ at the 3' end due to RNA Processing in nucleus

Poly-(A)

Explain the 4 stages of RNA transcription in prokaryotes: - Stage 1

Polymerase recognition of gene promoter

Bacterial - IF1

Prevents premature binding of tRNAs to A site

Hydroxylation of ___ and ____ is required for structural integrity and protein degradation

Pro;Lys

Gene Therapy - DNA Targets •Exons

Protein coding regions

Targeting proteins to endoplasmic reticulum - Step 5:

Protein synthesis resumes. The polypeptide passes through the translocon channel into the ER lumen. The signal sequence is cleaved by a signal peptidase and is degraded.

MicroRNA Processing • Binds ____ _____(with Argonaute proteins) where one ss-RNA is loaded • Recognizes and binds complementary sequence on mRNA(s) • Inhibits translation or cleaves and degrades mRNA

RISC complex

Hepatitis C virus (HCV) - Type: _____ - Family: Flavivrius - Cancers caused: Hepatocellular carcinoma

RNA

G1 phase (6-12 h)

RNA and protein synthesis. No DNA synthesis.

Reverse transcriptase is

RNA dependent DNA polymerase (transcribes RNA to DNA)

RNase H cleaves

RNA in an RNA/DNA hybrid

Therapeutic Potential of RNAi - Diseases linked to over-expression of genes or to gain of function mutations (Why?)

RNA interference is suppressing protein expression so if there is over expression you can use that. It there is a gain of function mutations that usually mean there is a mutation that causes the protein to have another effect besides its normal biological effect.

Retrotransposons need an _____ _____ for transposition.

RNA intermediate

In eukaryotes, TBP also involved in

RNA pol I & III complex assembly

Enhancers enhance initiation of transcription via binding specific transcription factors that impact

RNA pol II

In eukaryotes, binding of transcription factors to their DNA recognition sequences influences

RNA pol II activity and overall gene expression level

In eukaryotes, the TATA box is the critical sequence that allows

RNA pol II to recognize promoter

RNA pol III binds. Then transcription starts.

In absense of glucose, increase in cAMP. cAMP binds to Crp/CAP. Crp/CAP binds site upstream of the promoter of the Lac operon, helps recruit _____ ______.

RNA polymerase

In contrast, three different RNA Polymerases transcribe 3 different RNAs in eukaryotes (mammalian cells or viruses)

RNA polymerase I, RNA polymerase II, and RNA polymerase III

Death can be caused by inhibitors of RNA polymerase II - The toxin alpha-amanitin is an inhibitor of _________ and ________ , but not RNA pol I.

RNA polymerase II and RNA pol III

In positive regulation, a signal changes the conformation of an inactive activator, which then becomes active and binds to the regulatory region of a gene. Its presence aids binding of

RNA polymerase and helps "switch gene on"

DNA template ("antisense" strand) is copied by: - four-base language of DNA (A,T,G,C) is transcribed into four-base RNA language: A,U,G,C.

RNA polymerase to synthesize complementary RNA

Oncogenes and proto-oncogenes - First identified through their presence in _____ or _____. Hence designated by the prefix "v"-v-Src

RNA virus;retrovirus

Retrotransposons • Transcription to RNA (general cell machinery no special enzymes needed) • Reverse transcription of RNA to DNA - creates an ____________. • DNA synthesis from DNA strand to create a dsDNA molecule • Insertion into the host DNA site

RNA-DNA hybrid

Genome Replication: RNA viruses require viral __________ RNA polymerase.

RNA-dependent

Tumor viruses can cause cancer: - they can be ____ or _____ viruses

RNA;DNA

Reverse transcriptase is an

RNAse H

Gene Therapy with RPE65 • Leber cogenital amaurosis (LCA) due to functional loss of ______ (involved in phototransduction) • Subretinal injection of AAV.RPE65 vector • Clinical evaluation of vision recovery

RPE65

Information Flow: DNA --> RNA ---> Protein But can go from RNA --> DNA by what?

RT or RNA-dependent DNA polymerase

Most of the mRNAs are exported from the nucleus to cytoplasm by

Ran-independent transport mechanism

Pancreatic cancer has a point mutation in

Ras

Proto-oncogenes serve important cellular functions - Signal transducing proteins:

Ras, Abl

M cyclins: Cyclin B

Required for the entry into mitosis

G1/S Cyclins - Cyclin E

Required for the passage through the restriction point

Tumor Virus picked up an "oncogene" during an early replication cycle... - What is the concept?

Retroviral genome integrated into genome of host cell (perhaps a transformed cell?) ;RSV seen to have a "broken/activated" copy of a cellular gene (c-src) that is responsible for transformation --> 'v-src' gene picked up during viral evolution?

Prokaryotes: Initiation codes are preceded by a specific sequence called

Shine-Dalgarno sequence

Initiation of Prokaryotes - The correct binding of the fmet-tRNA^fmet to the P site in the 70S initiation complex is ensured by (2) ii. Interaction between the

Shine-Dalgarno sequence and the 16s rRNA (2)

Retrotransposons may be ancient or mutated retroviruses

Some of them even package their RNA into a virus like particle that never leaves the cell

Hallmarks of cancer - Develop angiogenic potential:

Stimulate formation of new blood vessels to bring nutrients and oxygen and to access the vasculature for metastasis

Biological Functions of miRNAs

Stress response, Hypoxia, Oncogenic signaling, Apoptosis and proliferation, and cell cycle control.

Viruses Classifications:

Structure: helical or spherical Genome: RNA or DNA | single/double stranded | sense/anti-sense) Enveloped / non-enveloped

(-)ssRNA animal viruses: Step 1?

Synthesis of (+) ssRNA

RNA synthesis in prokaryotes - Transcription

Synthesis of an RNA copy (mRNA, but also rRNA & tRNA) of information encoded by DNA genome

What is the fourth step in the life cycle of a typical virus?

Synthesis of viral proteins

(T or F) Streptomycin can cause miscoding and interferes with initiation.

(T or F): Apoptosis is vital to life

(T or F): Cancer cells can have more growth factors produced less dependent on the exogenous growth factor.

(T or F): Chaperones facilitate self-assembly process but do not convey additional information.

(T or F): Enhancers often consist of a cluster of DNA recognition sites, binding several proteins.

(T or F): Eukaryotic mRNAs contain a Cap and a Poly(A) tail

(T or F): alot of FDA Approved siRNA Therapies are being used

Eukaryotic gene control region (for mRNA coding genes): • promoter proximal elements occur within ~200 bp upstream of +1 site encompassing the

TATA box at -30.

Gene encoding reverse transcriptase subunit of telomerase ("TERT") is observed in brain cancers to have mutations in its promoter creating new binding sites for the specific transcription factor ETS --> leading to stronger initiation and elongation of _______ transcription and, therefore, more telomerase protein levels

TERT gene

What are the general transcription factors for RNA pol I?

TFI

What are the general transcription factors for RNA pol II?

TFII

What are the general transcription factors for class II genes?

TFIIA, TFIIB, TFIID (TBP + TAFs), TFIIE, TFIIF, TFIIH

• Release of RNA pol II from the promoter and elongation of the RNA requires three more TFII complexes: TFIIF, TFIIH, and TFIIJ. In particular, _________________.

TFIIH must phosphorylate the tail of RNA pol II before it can move.

What are the general transcription factors for RNA pol III?

TFIII

Is ECM cross-linking involved in TGFβ2-OHT? • Use Ad5.Cre in conditional Tgm2 ko mice • Use Ad5.TGFβ2 to induce OHT • KO of Tgm2 partially blocks

TGFβ2-OHT

Initiation in Eukaryotes Step 3:

The elF4F complex binds to mRNA

Targeting proteins to endoplasmic reticulum - Step 6:

The growing polypeptide extrudes through the translocon into the ER lumen until the complete peptide is synthesized.

Targeting proteins to endoplasmic reticulum - Step 4:

The peptide translocation channel (translocon) opens to accept the nascent polypeptide. The signal sequence is transferred to a hydrophobic binding site next to the central pore. GTP is hydrolyzed and SRP dissociates from the ribosome.

Elongation in Eukaryotes - Step One: eEF1α-GTP escorts the tRNA carrying the 2nd amino acid to the A site - Step 2: GTP is hydrolyzed and eEF1α is released - Step 3:

The peptidyl transferase activity of the large rRNA catalyzes peptide formation.

Elongation Step 3 in prokaryotes:

Translocation

Characteristics of apoptosis - Membrane asymmetry:

Translocation of phosphatidylserine to the outer leaflet of the plasma membrane

Eukaryotic Transcription of rRNA by RNA polymerase I • _____ protein binds both upstream control element and core promoter

UBF1

Contain dsDNA: Poxviruses (smallpox, cowpox, monkeypox):

Unlike other animal DNA viruses (which replicate inside cell nucleus), poxviruses replicate their dsDNA in the cytoplasm of the host cell.

In eukaryotes, Initiation of transcription:

Upstream elements facilitate initiation.

1. Lac operon = several genes encoding enzymes to help degrade lactose (e.g., lacZ encodes β-galactosidase) 2. When no lactose is available (or "gone"), Lac repressor (LacI) binds "operator sequence" which overlaps promoter of Lac operon. Thus the operon is turned off. 3.

When allo-lactose ("inducer") is present, it binds LacI and repressor is removed from operator. Operon turned on, starts transcription.

Gene Therapy - Ex Vivo

When cells are removed from an individual and genetic modification occurs on the outside of the individual in lab. Then the cells are given back to the individual.

1. Lac operon = several genes encoding enzymes to help degrade lactose (e.g., lacZ encodes β-galactosidase) 2.

When no lactose is available (or "gone"), Lac repressor (LacI) binds "operator sequence" which overlaps promoter of Lac operon. Thus the operon is turned off.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is an _______ disorder caused by misfolding and degradation of the enzyme.

X-linked

Hepatitis viruses (HBV, HCV) - Chronic infection (direct);

accompanied by chronic oxidative damage, promotes mutation and tumor development

(Eukaryotes) TFIIF

accompanies RNA Pol II as it binds to promoter

The ribosome - The small subunit provides a framework on which the tRNA can be

accurately matched to the codons of the mRNA

Retroviruses infect animals and includes the human immunodeficiency virus (HIV) that causes

acquired immunodeficiency syndrome (AIDS)

CAK kinase

activates CDKs

Cdc25 phosphatase

activates CDKs

Cdc14 phosphatase

activates Cdh1 to degrade mitotic cyclins

Eukaryotes - RNA pol II requires general transcription factors to assemble a pre-initiation complex to guide down, orient, and align RNA pol II in order to initiate transcription. - Assembled pre-initiation complex cannot normally initiate new transcription without the aid of upstream (and sometimes downstream) ___________. - Overall? Multiple steps of assembly prior to initiation and, therefore, multiple steps for speeding up or slowing down the process in response to regulatory proteins.

activator modules

Specific transcription factors in eukaryotes: _____ and ______ - only in specific tissues

activators and repressors

M-CDK phosphorylated at Thr-161 is ______.

active

Introduction to protein synthesis - tRNA =

adaptor

In negative regulation, a repressor protein blocks the gene's promoter. A signal changes the conformation of the repressor, releasing it from the gene and

allowing RNA polymerase to bind

- Eukaryotes use many more gene-regulatory proteins (activators, repressors, silencers, insulators, co-activators) ---> some are DNA-bound 100's to 1000's of bases away from (+1)! - Overall? Each eukaryotic promoter has potential to be controlled by multiple different regulatory DNA sequences scattered

along the chromosomal region of the gene locus

Eukaryotic pre-mRNA Alternative Splicing of Exons Produce Multiple mRNAs and Proteins - Eukaryotic cells can sometimes choose to use different splice sites within the same gene - Generally, ______ ______ is used by different cell types within the same animal

alternative splicing

In Vivo Gene Editing Therapy - LCA10 • LCA10 due to ___ ____ of CEP290/GUCY2D (guanidine cyclase in the vision transduction cascade) • 1st clinical example of in vivo CRISPR genome editing • Currently in phase III clinical trials (Editas)

alternative splicing

In response to an apoptotic stimulus, pro-apoptotic BH3-only proteins inactivate

anti-apoptotic Bcl-2 family proteins

DNA template

antisense strand

Eukaryotic gene expression can be controlled at _____ of six different stages including the transport of mature RNA from nucleus to cytoplasm.

any

Nuclear Localization Sequence may be located _______ along the primary sequence of the protein.

anywhere

Bcl-2 represents the first mammalian regulatory protein that inhibits

apoptosis

• The number of cells in multi-cellular organisms is tightly regulated. • Cell number is regulated not only by cell division but also by cell death. • Cell suicide or ______ is necessary to maintain a constant cell number.

apoptosis

Activation of initiator pro-caspase-9 - Cytochrome c facilitates interaction of apoptosis promoting activating factor (Apaf 1) with initiator procaspase-9 through CARD domain to form _____, causing activation of caspase-9.

apoptosome

Role of TGFβ2 in Glaucoma • Ad5.TGFβ2 transduction of mouse eyes • Increased TGFβ2 expression in _____ humor and ____ • TGFβ2-induced ocular hypertension • Target validation & new model

aqueous humor and TM

Glycosylation

attachment of carbohydrate side.

N-Myristoylation

attachment of myristic acid to an N-terminal Gly

(In prokaryotes) Tetracycline interferes with

attachment of tRNA to mRNA-ribosome complex

Positive regulation of prokaryotic gene transcription - Activator protein responds to an external signal (e.g., small molecule binding, or covalent modification) and then becomes

avid binder to a particular target DNA sequence

Ubiquitin-proteasome pathway - The proteasome has a large ___ like structure where the protein is degraded.

barrel

Nuclear Localization Sequence contains 4 to 8 amino acid residues and include several consecutive _____ residues.

bas (Arg or Lys)

Inhibitory Protein - CKIs p27^KIP1, p57^KIP2, p21^CIP

bind and inhibit CDKs

Cohesins

bind to DNA in S phase and maintain the linkage between sister chromatids

The product of the human papillomavirus oncogene E7 promotes cancer by

binding and preventing function of Rb protein

Elongation: Step 1 in prokaryotes -

binding of incoming AA-tRNA

Step 1 of initiation of prokaryotes:

binding of the mRNA to the 30S ribosomal subunit

Inhibitory Protein - Rb

binds E2Fs, preventing transcription of multiple cell cycle genes

Inhibitory Protein - INK4

binds and inhibits G1 CDKs

Inhibitory Protein - Sic1

binds and inhibits S phase CDKs

(Eukaryotes) TFIIB

binds downstream of TATA box; required for binding of RNA Pol II to promoter

Hepatitis viruses (HBV, HCV) - Viral proteins HBx (HBV) [or NS5B, NS2 (HCV)]

binds p53, disrupts normal growth control by transcriptional activation of several growth-promoting genes

(Eukaryotes) TBP

binds to TATA box, part of TFIID

(Eukaryotes) TFIIA

binds upstream of TATA box; required for binding of RNA Pol II to promoter

Adverse effects of Rifamycins?

body secretions turn orange

U2 binds the

branch site

Major Mechanisms for Protein Export and Import through the NPC - Proteins have a well-defined mechanism for export from and import into the nucleus • Nuclear export signal (NES) on • Ran-GTP dependent • Recycled import/export proteins

cargo proteins leaving nucleus

Extrinsic or receptor-initiated apoptosis - An initiator caspase can then cause activation of its downstream effector caspase, such as _____ and trigger caspase cascade.

caspase-3

Truncated Bid (tBid) translocates to the mitochondria and causes release of cytochrome c. Cytochrome c release causes activation of

caspase-9

Characteristics of apoptosis - activation of

caspases

Hallmarks of cancer - Resistance to

cell suicide or apoptosis

Characteristics of apoptosis - Expression of eat-me signal on the

cell surface

RNA polymerase II transcribes eukaryotic genes that produce mRNA and code for proteins - Recognition of the promoter and initiation of transcription by RNA polymerase II requires a number of general transcription factors expressed in every

cell type

In prokaryotes, the Shine-Dalgarno sequence is

centered 8-13 base pairs to the 5' side of the initiation codon.

Protein folding assisted by

chaperones

• Protein folding is assisted by a family of proteins called _______.

chaperones

Charging of tRNA -The aminoacyl group is esterified to the 2' or 3' position of the terminal adenylate residue. - The ester linkage activates the amino acid and joins it to the tRNA. It has a highly negative free energy of hydrolysis. - The coupling of each amino acid to its appropriate tRNA is called "___________."

charging of tRNA

p53 is induced by cellular stress such as DNA damage caused by

chemotherapeutic drugs, γ-irradiation, etc.

CAR (____ _____ _____)-T cell therapy

chimeric antigen receptor

Line elements and genome diversity • The LINE (RNA molecules do something!) • They aren't just being transcribed for no reason • They are bound to the nucleosomes and help establish _______ environment • They also create new insertions (in 1-20 to 1-200 live births) (rare) • Somatic retrotransposition has been associated with cancer • In the germline, deletions have been associated with sporadic inborn errors in metabolism.

chromatin

Barbara McClintock discovered transposable elements in corn. She noticed that despite very careful breeding of corn, the corn color was not uniform and could even differ between corn colors • Transposons in corn can change corn color • They can also cause ____ ____

chromosomal breakage

In Burkitt's lymphoma, myc proto-oncogene is converted to an oncogene by

chromosomal translocation

Oncogenic DNA viruses cause cancer by _____ ______. Tissue damage and/or stimulating cell division.

chronic infections

Proteins travel from the ER to the ____ ____ of the Golgi complex in transport vesicles.

cis side

What are the DNA sequences that specify transcription factor binding called?

cis-acting elements

Chromosome Segregation - Separase becomes activated and cleaves _______.

cohesin

Chromosome Segregation - Sister chromatids are held together by _____, a multi-protein complex

cohesin

The metaphase spindle - Sister chromatids are held together by multiprotein complexes called _______

cohesins

Line (L1) insertion into APC genes cause

colon cancer

Chaperone binding stabilizes the amino terminal portion in an unfolded conformation until the rest of the polypeptide is synthesized and the _______ protein can fold correctly.

completed

Transcriptional regulation in eukaryotes is more ______ than in prokaryotes

complex

In apoptosis, cell shrinks and chromosomes

condense.

The ras proto-oncogene can become an oncogene by a single point mutation that alters its protein product to have ____ activity.

constitutive Raf-activation

Enhancers "_____" theRNA pol II transcriptional assembly via specific transcription factors.

contact

Formation of pre-initiation complex at ___ ____ by general transcription factors and RNA polymerase II

core promoter

Gene Therapy - Genome Editing •Prime editing (________)

correct and/or replace disease gene

Stage 3: Elongation The nascent polypeptide is lengthened by _______ attachment of successive amino acid units.

covalent

Methylation

covalent addition of methyl groups

Reverse Transcriptase - DNA dependent DNA polymerase:

creates double stranded DNA from single stranded DNA

Eukaryotes • Recognition is due to base pairing between the snRNA and the primary transcript • The protein part of the SNURP supervises the

cutting and joining reactions

Cell cycle progression requires

cyclical activation of cyclin-dependent protein kinases (CDKs)

CDKs are serine/threonine protein kinases; require _____ for activity. The activity of these kinases rises and falls as cells progress through the cell cycle.

cyclin

What cyclin is the only growth factor sensor?

cyclin D

DNA damage checkpoint detects damaged DNA and arrests cells to allow repair of the

damaged DNA

In DNA transposons, sometimes the host DNA is incorporate rather than the transposon. The insertion sequences stick to the host DNA rather than the transposon, and this DNA is inserted into the recipient. This is ____ _____ process as there is no transposase in the host DNA.

dead-end

Extrinsic or receptor-initiated apoptosis - Binding of initiator caspase (e.g., caspase-8) to DISC (_______) causes autoproteolytic activation.

death-inducing signaling complex

Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator). The mutation causes

defective protein folding and reduced levels of functional protein

Apoptosis provides a _____ _____ in the adult. - eliminates redundant, damaged, and infected cells.

defense mechanism

Wobble Hypothesis • The first two bases of the mRNA codon form strong Watson-Crick base pairs with the corresponding bases of the tRNA anticodon. • Base-pairing in the third codon base is variable (_______). • The first base of the anticodon is called the Wobble base. • The first base of the anticodon (reading in the 5' → 3' direction) determines the number of codons recognized by the tRNA.

degenerate

Human Papilloma Virus (HPV) - E6 binds to p53

degradation by proteosome

Ubiquitin-Protein Ligases: SCF

degradation of phosphorylated Sic1 or p27^KIP1 to activate S phase CDKs

Ubiquitin-Protein Ligases: APC/C^Cdc20

degradation of securin, initiating anaphase. Induces degradation of B-type cyclins

Inactivation of p53 and Rb by tumor viruses - E6 targets p53 and ______ it.

degrades

Components of Lac operon Single lac mRNA is translated to produce: - LacZ (β-galactosidase) --->

degrades lactose

Step 3 of initiation of prokaryotes: formation of initiation complex - (3) All initiation factors (IF1, IF2, and IF3)

depart from the ribosome

Phosphorylation of lamins causes _______ of lamin filaments.

depolymerization

Apoptosis removes unwanted cells during

development

Biological Roles for RNAi •Involved in regulating expression of at least 1/3rd of all protein coding genes (>2000 miRNAs) • Roles in _________ • Roles in wide variety of diseases • Biomarkers for disease • Potential new therapeutic agents • Research tools

development

Defects in ubiquitin proteasome pathway contribute to many ______.

diseases

Human Papilloma Virus (HPV) - E7 binds to pRb

displacement of E2F transcription factor (activation of genes); also inactivates CDK inhibitor p21, activates Cyclin E and A.

Restriction point is the point where decision to _____ takes place.

divide

Human Papilloma Virus (HPV) - needs _______ host cells

dividing

Initiation of transcription: Upstream elements close to promoterbind specific transcription factors - Upstream elements bind to specific transcription factors to increase efficiency of initiation by RNA pol II; these factors contact TFIID, TFIIB, TFIIA, etc. and help complex assembly but

do not directly contact RNA pol II

Sine Elements • These are non-autonomous, which means they _______________. • Contain a SRP - signal recognition particle • Internal RNA polymerase III promoter • Poly A-rich tail

do not work without LINE elements providing the necessary proteins

Classification of Viruses - Class I:

double stranded DNA (human HSV, prokaryotic virus T4)

Classification of Viruses - Class III:

double stranded RNA (+ strand & - strand RNA; Reoviruses like rotavirus)

In eukaryotes, enhancers might be upstream, might be one or two or ten or twelve or might be

downstream

U4 and U6 bind to U2, and U5 binds to the

downstream exon

Human Papilloma Virus (HPV) - small circular ______ (episomes)

dsDNA

Classification of Viruses - Class VII:

dsDNA viruses with an RNA intermediate reverse transcribed in its lifecycle (human hepatitis B virus [HBV])

(+) ssRNA animal viruses - Retroviruses (enveloped)

e.g., HIV (Human immunodeficiency virus), HTLV (Human T-cell lymphotropic virus)

(+) ssRNA animal viruses - Flaviviruses (enveloped)

e.g., Yellow fever, dengue fever, West Nile virus, Hepatitis C virus (HCV)

Elongation in Eukaryotes - Step 1

eEF1α-GTP escorts the tRNA carrying the 2nd amino acid to the A site.

Termination in Eukaryotes - Translation is terminated when termination codons (UAA, UAG, or UGA) are at the A site. - The release factor ____ bound to GTP escorts ____ to the A site.

eRF3; eRF1

In Liddle syndrome, a sodium channel in the kidney is not degraded causing excessive Na+ absorption and

early-onset of hypertension

Stage 3: Elongation - Efficiency and fidelity of elongation are enhanced by non cytosolic proteins called "___ ____ (EF)."

elongation factors

Elongation in Eukaryotes - it is similar to prokaryotes - it requires:

elongation factors eEF1α (EF-Tu) and eEf2 (EF-G)

Human Papilloma Virus (HPV) --->

epithelial cancers

Small molecule inhibitors of EGFR (______, _______ and _______) are used in the treatment of cancers in which EGFR is activated, such as lung cancers.

erlotinib, lapatinib, and gefitinib

Oligosaccharides are added to the C-terminal carboxyl groups of some proteins by an

ethanolamine

These antibiotics often inhibit protein synthesis in bacteria but are relatively harmless in ________.

eukaryotes

- Several general transcription factors (such as TFIIA,IIB, etc.) are required for expression of eukaryotic protein coding genes. - Expression also requires specific transcription factors that only affect certain genes in response to specific stimuli or signals. - Eukaryotic transcription factors may bind to upstream elements in promoter region or to enhancer elements ______ ______ from promoter.

far away

Eukaryotic gene control region (for mRNA coding genes): Eukaryotic gene regulatory proteins can control transcription (turn on or off) when bound to DNA

far away from promoter

Summary of RNA pol II considerations for mRNA transcription: Controlled by b) DNA-bound regulators

far removed from the start of transcription

Protein disulfide isomerase (PDI) stabilizes the

folded structures by catalyzing the formation of disulfide bonds between cysteine residues

Chaperonins, a class of chaperones, aid

folding of large proteins.

Step 3 of initiation of prokaryotes:

formation of initiation complex

Step 2 of initiation of prokaryotes:

formation of pre-initiation complex

Elongation Step 2 in prokaryotes: peptide bond formation - The carboxyl end is released from the tRNA and attaches to the _________ of the AA-tRNA at the A site.

free NH2 group

Pre-mRNA Splicing and Pathogenesis of Human Diseases - Disease: β-Thalassemia • Point mutations in 5' splice site, 3' splice site, or branch point 'A' may not produce ____ ______ mRNA or protein causing the genetic disease beta-thalassemia

functional β-globin

What are the three major genes in the HIV genome?

gag, pol, and env

Apoptotic cells produce "eat me" signals, which include _______ of cell surface _______.

gain;phosphatidylserines

Some prokaryotic promoters function poorly or not at all in the absence of extra proteins known as

gene activator proteins

What are the creation of pseudogenes?

gene duplications

RNA Interference • Double stranded RNA (19-28 nt) interferes with ______ ________ - > 28 nt ds RNA elicits innate immune response

gene expression

Enhancers are DNA sequences involved in ___________, especially during development or in different cell types.

gene regulation

HAT activates

gene transcription

Summary of RNA pol II considerations for mRNA transcription: Controlled by a) ________ (TFII-A, -B, -D, -E, -F, -H) and ______ ("pre-initiation complex") at core class II gene promoter

general transcription factors ; RNA pol II

RNA Interference • Targets specific mRNAs based on sequence homology • microRNA (miRNA):

generated from endogenous RNA

The _____ _____ is the relationship between the sequence of bases in the DNA or mRNA and the sequence of amino acids in proteins.

genetic code

Viruses in the bacterial (prokaryotic) world - Bacteriophages leave their protein shell behind and only their ______ enters the host bacterium.

genome

• Most transposons in the human genome are silent and do not move • Transposons drive ____ ____

genome evolution

Structural features of tRNA - Single-stranded RNA chains (73-93 nts) - Complementary base-paring of four segments with single-stranded open loop regions-looks like a clover leaf -Most tRNAs have a _____ (pG) residue at the 5' end. - The trinucleotide CCA at the 3' end is the amino acid binding site. - The anticodon loop at the opposite side recognizes appropriate codons by complementary base pairing.

guanylate

Alu insertion into factor VIII gene causes

hemophilia

Hepaptitis B virus (HBV) --->

hepatocellular cancers

Hepatitis viruses (HBV, HCV) - major etiological agents of

hepatocellular carcinoma

Normal Cell Division + Normal Apoptosis =

homeostasis

Chromosomal translocation - Formation of ______ genes by recombination of two unrelated chromosomes can activate growth-promoting gene products

hybrid

Stage 4: Termination and release • Completion of the peptide chain is signaled by termination codons UAG, UAA and UGA. • Cells do not contain tRNAs with anticodons complementary to these termination signals. • Release factors (RF) recognize the signals and

hydrolyze the terminal peptidyl-tRNA bond to terminate protein synthesis

Step 3 of initiation of prokaryotes: formation of initiation complex - (2) GTP bound to IF-2 is

hydrolyzed

oncogenic kinase active

hyperactive oncogenic kinase + activating phosphate = signal for cell proliferation and survival ---> leukemia

Match the inhibitors of protein synthesis with their mechanisms of action. i. Inhibits elongation in eukaryotes. ii. A potent inhibitor of protein synthesis that acts as an analogue of aminoacyl-tRNA. iii. Inhibits binding of aminocyl-tRNA to A site. iv. Binds to small rRNA v. Inhibits peptide bond formation in prokaryotes. A. Choramphenicol B. Puromycin C. Cycloheximide D. Tetracycline E. Streptomycin

i. C ii. B iii. D iv. E v. A

What shape is the adenovirus?

icosahedral

Prokaryotic Promoter "strength" depends partly on closeness to

ideal consensus -10 and -35 sequences (Strongest promoters are often identical to consensus)

Oct-2 found only in _______ (helps activate genes encoding antibodies)

immune system cells

Major Mechanisms for Protein Export and Import through the NPC - Proteins have a well-defined mechanism for export from and import into the nucleus • Nuclear localization signal (NLS) on • Ran-GTP dependent • Recycled import/export proteins

import cargo into nucleus

Retrotransposons and Disease • Deletions - Through activity they can cause deletions • They can also cause deletions based on the duplicated nature of the sequences • Point mutations:

in and around insertion sites

Oncogenic viruses can bind to and

inactivate Rb

p53 is frequently ________ in many cancers

inactivated

oncogenic kinase blocked with Gleevec

inactivated oncogenic kinase = no signal --> no leukemia

Inactivation of p53 and Rb by tumor viruses - E7 binds to Rb and _______ it.

inactivates

In normal cells, pro-apoptotic BH3 only proteins are _______.

inactive

Transposons for gene therapy - Potential for use in _____ _____ of metabolism and other Mendellian diseases.

inborn errors

(Eukaryotes) TFIID = TBP + TAFs

includes TBP, recognizes Pol II specific promoter

Stage 3: Elongation A peptide bond is formed between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an

incoming amino acid

In absense of glucose, ____________. cAMP binds to Crp/CAP.

increase in cAMP

miRNAs in Cardiac Ischemia - affecting the regulation of number of proteins being expressed. - miRNA's can be increased which means silencing of the protein. - miRNA can be decreased which means you get

increase of gene expression of that protein

Latent/latency - Viral infection does not kill host cell. - Can be "productive" (produce viral particles) or "non-productive" (no viral particles are produced) - Viral genome integrated into the host genome. - Latent viruses can be activated after months/years of dormancy - Infected cells can be transformed and ____ ____ in animals (tumor viruses)

induce tumors

The enzyme β-galactosidase splits lactose into galactose plus glucose. β-galactosidase can also interconvert lactose with its isomer, allo-lactose, which is the true _____ of the lac operon.

inducer

Mutations in the p53 gene are the most common aberrations in human cancers. Wildtype p53

induces cell cycle arrest or apoptosis in response to DNA damage

p53 causes tumor suppression by

inducing apoptosis

The life cycle of a virus alternates between two forms:

inert virus particle (virion) and an active intracellular stage

Human Papilloma Virus (HPV) - HPV16, HPV18:

infect cells of squamous epithelia (skin, mucous membrane); viral genome seen in 85% of invasive squamous cell cervical carcinoma

Many bacterial pathogens can hijack eukaryotic ubiquitination system to eliminate host proteins to facilitate ______.

infection

In necrosis, swelling of the ER and mitochondria and breakdown of plasma membrane, organelles, and nucleus; leakage of contents. Causes

inflammation

Bcl-2 promotes cancer by

inhibiting apoptosis

The dual-specificity Wee1 kinase phosphorylates _______ phosphorylation sites

inhibitory

Wee1 kinase

inhibits CDKs

Epstein Barr Virus (EBV) - vIL10: viral cytokine

inhibits T-cell immune response

Eukaryotic DNA-dependent RNA polymerase II cannot

initiate transcription on its own

Eukaryotic RNA Polymerase III transcribes genes from a Class III gene promoter - Internal Promoter for RNA pol III: Gene (e.g., for 5S rRNA, for tRNA)

is transcribed using a promoter located within the gene itself (recognition sites are downstream of transcription start site +1)

Peptidyl proline isomerase catalyzes the

isomerization between the cis and trans configurations of prolyl-peptide bonds

Genome Editing - Nuclease Specificity • Overall efficiency of modifying specific target DNA ("on-target") • What does "off-target" mean?

it can cleave a variety of other sites that are not being specified by the guide strand

CRISPR/Cas9 Gene Therapy in MYOC Glaucoma •No phenotype in MYOC null humans and mice •Current function of MYOC not known • Generation of transgenic hMYOC.Y437H mouse • CRISPR/Cas9 to ______ MYOC gene •Prevents and rescues MYOC glaucoma in mice - Lowers IOP - Improves retinal function (PERG)

knock-out

Components of Lac operon Promoter lacP is regulated by binding of repressor at the operator, ____, (and by binding of Crp protein at Crp site)

lacO

Components of Lac operon lacZ, lacY, and lacA all transcribed from a single promoter, designated ______.

lacP

R-Loop Analysis Experiment Visualizes Introns - Electron microscopy has been used for direct visualization of eukaryotic introns - Both DNA and mRNA contain the exons that comprise the coding sequence, but the final mRNA _____ the introns (non-coding regions)

lacks

Components of Lac operon Single lac mRNA is translated to produce: LacY --->

lactose permease/ transport protein

What is an example of negative regulation?

lactose responsiveness

Nuclear envelope breakdown - phosphorylation of ______ by M-Cdk (cyclin B/Cdk1)

lamin

Nuclear envelope is maintained by highly organized meshwork of intermediate filaments composed of ______.

lamins

Epstein Barr Virus (EBV) - Infection is

latent (no virus replication, no cell death)

Altered Expression of Spliceosome Proteins in NTM and GTM Cells Changes GRα/β Expression - GTM cells have lower expression of GRβ , making them more susceptible to damaging effects of GCs - Altered expression of spliceosome proteins regulates ?? - Decreased expression of SRp40 in GTM cells favors GRα splicing, making GTM cells more sensitive to GCs

levels GR alternative splicing

Initiator caspases contain a

long pro-domain

The HIV genome is flanked by two _____ _____ ____ (LTR's) required for insertion of the dsDNA version of virus genome into host chromosomal DNA.

long terminal repeats

In prokaryotes, ribosomes translate mRNA in the 5' to 3' direction, the ribosome closest to the 3' end has the

longest nascent peptide

Genome Editing - Nuclease Specificity - Therapeutic index

looking to get the best clevage of the specific target and is not allowing off target effects.

Epstein-Barr virus (EBV) --->

lymphoid malignancies (B, T cell lymphoma)

Ubiquitin, a short peptide of ~76 amino acid residues, attach to _____ residues of proteins and mark them for degradation.

lysine

Autophagy - cellular stresses, such as nutrient deprivation, active autophagy genes, which intiate the formation of membrane-bound vesicles in which cellular organelles are sequestered. These vesicles fuse with ________, in which organelles are digested, and the products are used to provide nutrients for the cell. The same process can trigger apoptosis by mechanisms that are not well defined.

lysosomes

Protein sorting to

lysosomes, plasma membrane, and secretory vesicles

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA - PABP also binds

m7G cap end, protecting it from being cut off

Decoding mRNA by transfer RNA (tRNA) - ____ carries the genetic information transcribed from DNA

mRNA

RNA pol II transcribes what?

mRNA

Initiation in Eukaryotes Step 3: The elF4F complex binds to mRNA - eIF4F complex: - elF4E binds to

mRNA 5' cap

RNA pol II in eukaryotes need general transcription factors to initiate

mRNA transcription

Step 2 of initiation of prokaryotes: formation of pre-initiation complex - (3) The anticodon of this tRNA now pairs correctly with the

mRNA's initiation codon

One the viral DNA or RNA enters the cell, the virus uses host cell to manufacture more copies of viral genome and to

make the proteins for assembly of new virus particles

Acetylation of Lys on histones neutralizes its positive charge and

makes DNA less compact

Human Papilloma Virus (HPV) - HPV6, HPV11:

malignant potential low; genital warts

Defect in apoptosis is associated with

many diseases

In the HIV genome, gag =

matrix and capsid protein

5'-Capping (in the nucleus) is first step in eukaryotic mRNA _______

maturation

- DNA folding of allows numerous activators (bound to enhancer sequences) to approach the transcription apparatus. - The _____ _____ allows information from the activators (and/or repressors) to influence RNA pol II activity.

mediator complex

Viruses vs Cells: - Living cells have ______, whereas most viruses do not (or massively crowded w/ protein).

membranes

Hallmarks of cancer - Reprogram energy

metabolism

Translation initiates with the amino acid ______, encoded by ______ near the 5' end of the mRNA.

methionine;AUG

After GTP addition, guanine base has a ______ group attached at 7-position. Known as "cap0" or "m7G".

methyl

Dicer dependent:

miRNA and siRNA

Regulation of DNA damage-induced apoptosis by tumor suppressor protein p53 - It promotes apoptosis via the ______ pathway

mitochondrial

Bcl-2 family proteins regulate

mitochondrial integrity

p53 promotes apoptosis via the

mitochondrial pathway

Activation of M-CDK is required for entry into _____.

mitosis

What are the CDK-1 (cyclin A & B) function?

mitosis

M phase (1 h)

mitosis (nuclear division) and cytokinesis (cell division) yields two daughter cells.

M phase contains what two processes?

mitosis and cytokinesis

In contrast to prokaryotic transcription, one structural gene produces one ________ RNA which is translated to produce one single protein in eukaryotes

monocistronic

Herceptin (trastuzumab), a humanized _______ antibody against HER2 is the first targeted biologic therapy for breast cancer. Herceptin also potentiates sensitivity of chemotherapeutic agents.

monoclonal

Sine Elements - Alu Repeats - ALU accounts for about 11% of the human genome sequence - ALU elements produce _________. - Each repeat ~ 280 bp long

most of the disease causing insertions in humans

Hereditary retinoblastoma - inherited mutant Rb gene - occasional cell inactivates its only good Rb gene copy --> excessive cell proliferation leading to retinoblastoma - Result: ???

most people with inherited mutation develop multiple tumors in both eyes

One gene can encode

multiple proteins

Diseases can be caused by ______ within promoter DNA sequences

mutations

number of exons in the gene (n) = __________

n+1

Binding of a cyclin is _____ but not sufficient.

necessary

In _____ regulation, a repressor protein blocks the Lac operon. Lactose binding changes the conformation of the repressor LacI, releasing it from the gene and allowing RNA polymerase to bind.

negative

During mRNA elongation, RNA pol II is also subject to

negative regulation

Addition of Poly (A) tail is the second step in maturation of eukaryotic mRNA - poly(A) polymerase adds 100~200adenine residues, and PABP remains bound to

new 3' poly(A) end

The life cycle of a virus 3. (Uncoating and) replication of viral genome 4. Synthesis of viral proteins - Viruses do not 'divide' like cells, but instead their genomes encode protein components required for

new virion assembly

Cell cycle checkpoint monitors orderly and accurate transitions of one phase of the cell cycle to the =

next phase

The HIV genome is very compact and only has _____ genes.

nine

Double-Stranded RNA (dsRNA) Viruses - relatively uncommon; reovirus are best known. - Spherical: two concentric protein shells but __ ____. • Contain ~12 double-stranded RNA molecules, each coding for a single virus protein.

no envelope

Normal Healthy Individual - occasional cell inactivates one of its two good Rb genes - Result: ????

no tumor

p53 is highly unstable and is present at very low concentration under ______ condition

normal

Neoplasia is a process(es) by which the

normal controlling mechanisms that regulate cell proliferation, differentiation, cell death and senescence are impaired, resulting in progressive growth.

Tumor supressor genes

normally encode proteins that inhibit proliferation and promote cell death

Proto-oncogenes

normally promote cell survival or proliferation

Genome maintenance genes

normally repair or prevent damage

Unlike retroviruses, oncogenic DNA viruses usually do _____________ into host genome (have "episomal existence"); no proto-oncogene activation

not fully integrate

• Transport of macromolecules including mRNPs, tRNAs, rRNAs out of the nucleus as well as the transport into the nucleus of all nuclear proteins synthesized on ribosomes in the cytoplasm occurs via ___________, which are much different than transport mechanisms for small molecules and ions across cellular membranes • Large and small molecules enter and leave the nucleus via Nuclear Pore Complexes (NPC)

nuclear pore mechanisms

Gene Therapy - Genome Editing - CRISPR/Cas genome editing • Cas = _______ • DNA target requires PAM sequence • Guide RNA to specifically bind to target DNA

nuclease

Backwards connection stabilizes mRNA --> protects against mRNA degradation by _______.

nucleases

The Nuclear Pore Complex (NPC) • Numerous pores are elaborate complex structures that perforate the nuclear envelope • NPC is one of the largest protein assemblies (60-80 million Daltons) in the cell • NPC contains 30 different proteins called _________.

nucleoporins

After all processing is complete, the mRNA exits the _____ to be translated by ribosomes

nucleus

Protein targeting to nucleus • Nuclear localization sequence (NLS) targets a protein to the ___.

nucleus

Spliceosome in eukaryotes consists of several proteins and RNAs • Some specialized, small RNA molecules found only in the ______.

nucleus

Number of R-loop (n)=

number of introns

Where does protein sorting occur of the Golgi Complex?

occurs in the trans side

For using these other sugars (such as lac operon for lactose ) need to be turned on. When glucose is available, genes for using other sugars switched ____.

off

Extrinsic or receptor-initiated apoptosis - Binding of death ligand to the receptor triggers receptor _____

oligomerization

_________: Promote cell growth in the absence of growth-promoting signals

oncogenes

Epstein-Barr virus (EBV) is an

oncogenic herpes virus

p53 targets _____ _____ SV40, HPV, adenovirus, etc.

oncogenic viruses

In inherited retinoblastoma, _____ defective copy is genetically transmitted

one

Eukaryotes have monocistronic genes (each gene transcribed separately from individual promoters) VS. Genes often transcribed as a polycistronic mRNA transcript:

one long transcript encoding several protein ORFs

Initiation of transcription in prokaryotes - Polymerase/DNA forms tight complex, melts DNA --> forms an

open complex

ORF = _____________ - In theory, any DNA or RNA sequence that could encode a protein if translated 5'-> 3' in triplet (codon) groups (no interrupting stop-codons)

open reading frame

Allo-lactose (or IPTG) binds to the constitutively synthesized repressor which prevents its binding to the ______ and prevents repression. Transcription of the lac operon starts. The operon is on.

operator

Several polycistronic genes (collectively called "______") are controlled in their simultaneous transcription as a single polycistronic mRNA off a "single operator site" at one regulatory gene locus in prokaryotes

operons

Chromosome Segregation - Separase breaks down cohesion and allows the sister chromatids to move to _______ spindle poles.

opposite

Enhancer DNA sequences can work in either

orientation

The HIV genome has several genes _____ in sequence with each other and are shown side-by-side in the diagram.

overlap

Eukaryotic pre-mRNA Alternative Splicing of Exons Produce Multiple mRNAs and Proteins - This allows a single original DNA sequence to be used to make several different proteins that have distinct but ______ functions - However, selection of which alternative splice site to use in a particular cell or tissue must itself be controlled, and this often requires additional proteins

overlapping

The amplification of DNA sequences of a proto-oncogene can cause

overproduction of the protein

lacI gene (encodes LacI repressor) has its ___________ and is transcribed in the direction opposite to the lacZYA operon

own independent promoter

Activation of Cyclin E-Cdk2 - In Go and early G1, Cyclin E-Cdk2 or G1/S-Cdk is associated with Cdk inhibitor ______.

p27

Activation of Cyclin E-Cdk2 - Growth factor signaling inhibits

p27 synthesis

Regulation of DNA damage-induced apoptosis by tumor suppressor protein

p53

History: Are viruses the causative agents for many or all human cancers? 1909: Peyton Rous discovers chicken sarcoma virus (Rous Sarcoma Virus or RSV) that can transform cells in culture! 1950's : DNA viruses senn inducing cancer (HPV --> ___________) in rabbits.

papillomas / warts

Proteolytic processing • Cleavage of signal sequence • A short sequence at the amino terminal end of certain proteins • Cleaved by a _______ during translation or after the protein reaches its destination

peptidase

The ribosome - The large subunit catalyzes the formation of the _____ ______ that link the amino acids together into a polypeptide chain.

peptide bonds

Elongation Step 2 in prokaryotes: peptide bond formation - This peptide bond formation is catalyzed by the ______ _____ activity of the 23S rRNA (ribozyme) in the large ribosomal subunit.

peptidyl transferase

Capping reaction uses three separate enzymes: FIRST

phosphatase removes 5' phosphate from transcript

Translocation of ________ to the outer leaflet of the plasma membrane for apoptosis.

phosphatidylserine

Activation of Cyclin E-Cdk2 - When Rb releases E2F and Cdk2 becomes activated, it _______ p27.

phosphorylates

(Eukaryotes) TFIIH

phosphorylates the tail of RNA Pol II, retained by polymerase during elongation

Disease Based IOP Lowering in SFRP1-Induced OHT • Increased expression of SFRP1 in GTM • Validation of SFRP1/WNT β-catenin signaling regulating IOP • Levels of β-catenin regulated by ______ (via GSK3b) • Topical ocular administration of GSK3b inhibitor reversed SFRP1-induced ocular hypertension

phosphorylation

Mitotic protein kinases - Chromatin Condensation:

phosphorylation of condensins and cohesions

Mitotic protein kinases - Nuclear Envelope Breakdown:

phosphorylation of lamins, nuclear pore complexes, and inner nuclear membrane proteins.

Regulation of M-CDK by

phosphorylation-dephosphorylation

Clinical Ex Vivo Example - iPSC Therapy • Generate iPSCs from patient's fibroblasts • Genome editing to correct mutation • Differentiate iPSCs into retina cells (e.g. _____ & ____ ____) • Subretinal implantation of engineered cells • Ongoing clinical trials

photoreceptors; retinal pigmented epithelial cells

The cyclic assembly, activation and disassembly of cyclin-CDK complexes are the _____ _____ during the cell cycle.

pivotal events

The hydrophobic lipid facilitates targeting and anchoring proteins to the

plasma membrane

Step 1 of initiation of prokaryotes: binding of the mRNA to the 30S ribosomal subunit - (1) IF-3 prevents

premature association of 30S and 50S subunits

Initiation in Eukaryotes Step 1: - (2) elF3 prevents

premature association of the ribosomal subunits

Step 1 of initiation of prokaryotes: binding of the mRNA to the 30S ribosomal subunit - (2) IF-1 prevents

premature binding of tRNA to the A site

What is the aggregating protein in Spongiform Encephalopathies?

prion protein

A rare transmissible neurodegenerative diseases, called spongiform encephalopathies (e.g., scrapie, mad cow disease, kuru) are caused by misfolded proteins _____ that are capable of self-replication.

prions

Cell death is regulated by

pro- and anti-apoptotic Bcl-2 family proteins

Activation of initiator pro-caspase-9 • Caspase-9 activates its downstream effector caspases, such as _______ and triggers caspase cascade, ultimately causing cell death.

pro-caspase-3/-7

Mitochondrial or intrinsic cell death pathway - The major mechanism of apoptosis triggered by various physiological and pathological stimuli. - Mitochondrial integrity is compromised causing release of _____ ______.

proapoptotic molecules

Other causes of Thalassemia • Mutations in the promoter region of the β-globin gene may not transcribe the gene and may not

produce β-globin

Latent/latency - Viral infection does not kill host cell. Can be "______" (produce viral particles) or "_______" (no viral particles are produced)

productive;non-productive

Role of TGFβ2 in Glaucoma • Glaucoma leading cause of vision loss and blindness •TGFβ2 = ????

profibrotic cytokine

Reverse transcriptase is NOT good at what?

proof reading and is error prone

After the protein is transported to the mitochondria, that targeting sequence is removed by

protease

Viruses are packages of genes inside

protective protein shells

Nuclear Localization Sequence is not removed after

protein at the nucleus

Retroviruses (not carrying an oncogene) can also cause cancer by integrating next to cellular "_______" (c-onc) and increasing its expression beyond normal limits

proto-oncogene

Mutated form of normal cellular genes are called

proto-oncogenes

Oncogenes and proto-oncogenes - Cellular counterparts or _____ are designated by the prefix "c"- c-Src

proto-oncogenes

Point mutation in Ras is the most common abnormality in

proto-oncogenes in human

RNA pol I transcribes what?

rRNA

Resolvase

recognizes internal resolution site (IRS)

Transposase

recognizes the inverted repeats and target sequences

Protein misfolding may lead to _____ _____ of a functional protein.

reduced levels

Prokaryotes - "In front" of each gene is a _______ DNA sequence that is not transcribed. RNA polymerase binds to the sequences within the promoter

regulatory

Chromosomal translocation can cause overproduction of a protein by placing them under the ____ ____of immunoglobulin gene.

regulatory element

In the HIV genome, rev =

regulatory function

Termination in Prokaryotes • Termination is triggered by the stop codons UAA, UAG and UGA at the A site. • The ____ _____ (RFs) bind to the termination codon at the A site.

release factors

Truncated Bid (tBid) translocates to the mitochondria and causes

release of cytochrome c

Effector pro-apoptotic Bcl-2 family proteins become activated causing

release of mitochondrial cytochrome c

Initiation in Eukaryotes Step 5: - GTP is hydrolyzed and most of the eIFs are

released

released from the ribosome

Modifications of individual amino acids - Phosphorylation / Dephosphorylation • Protein kinases add phosphate to the hydroxyl groups of Ser, Thr and Tyr • Alters the activity of a protein • Protein phosphatases ___ _____ • Function - Signal transduction - Metabolism - Cell cycle - Cell death

remove phosphate

Double-Stranded RNA (dsRNA) Viruses - Relatively uncommon; ______ are best-known.

reoviruses

Gene Therapy - Genome Editing •Base substitution (_____)

repair

Gene Therapy - Genome Editing •Gene knockin (_______)

replacement

Interphase is chromosome

replication

Repressors can also ____ transcription at a distance

repress

Passage through the restriction point - Rb associates with E2F and _____ its transcriptional activity.

represses

Bacterial transposon also encodes a _________ for transposition which keeps the transposon from jumping: it does this via a frame shift

repressor

Some prokaryotic promoters function poorly or not at all in the absence of extra proteins known as gene activator proteins. Also, an opposing class of gene regulator proteins known as ______ turn genes off.

repressors

Active intracellular stage of a virus

reprogramming the host for virion production

S cyclins - Cyclin A

required for initiation DNA replication

(Eukaryotes) TFIIE

required for promoter clearance and elongation

(Eukaryotes) TFIIJ

required for promoter clearance and elongation

Replicative transposition requires an additional enzyme

resolvase

Activation of Cyclin E-Cdk2 is required for passage through the

restriction point

Elongation of mRNA chain: - When DSIF and NELF are phosphorylated, along with carboxy-terminal domain (CTD) of RNA pol II, transcription can ____. - Majority of genes have "paused RNA pol II" due to unphosphorylated DSIF & NELF; thought is that initiation occurs frequently, but productive transcription occurs only upon need for gene product signaled by phosphorylation.

resume

First tumor suppressor gene identified in ______, a childhood eye tumor

retinoblastoma

Human T-Cell Leukemia Virus (HTLV-1) - only human ______ implicated in cancer pathogenesis

retrovirus

Human T-cell leukemia virus (HTLV-1) - Type: RNA - Family: ______ - Cancer caused: T-cell leukemia

retrovirus

A different kind of transformation - RNA Tumor Viruses - rate of transformation slower vs.

retrovirus-with-oncogene (v-onc)

In the HIV genome, pol =

reverse transcriptase, intergrase, protease

The spliceosome in eukaryotes consists of several ________ , also known as "SNURPS"

ribonucleoproteins (U1 to U6)

Initiation of Prokaryotes - The correct binding of the fmet-tRNA^fmet to the P site in the 70S initiation complex is ensured by (3) iii. Binding interactions between the

ribosomal P-site and the fmet-tRNA^fmet (3)

Viruses vs Cells: Living cells possesses their own _______ to make proteins.

ribosomes

1st-line Tuberculosis drug class - Rifamycins

rifapentine, rifampicin / rifampin

Addition of Poly (A) tail is the _____ step in maturation of eukaryotic mRNA

second

Amino acid activation • High degree of specificity of the aminoacyl-tRNA synthetases is necessary for correct translation of genetic messages. • The proofreading ability of aminoacyl-tRNA synthetases enhances the fidelity of protein synthesis. • If, however, an incorrect amino acid is attached to a tRNA, there is no mechanism during translation to detect the error and the incorrect amino acid will be incorporated into a protein. • The interaction between aminoacyl-tRNA synthetases and tRNAs has been referred to as the "____________."

second genetic code

CRISPR/Cas9 Gene Therapy in MYOC Glaucoma • Ex-vivo perfusion culture of human eye anterior segments • Use same CRISPR/Cas9 gene delivery to cultured eyes • Access MYOC knock-out by looking for ____ _____ in perfusate (E) and mRNA in tissue (F) • Challenges to moving into clinic

secreted myocilin

Chromosome Segregation - Separase is inactive when bound to a protein called _______.

securin

DNA coding strand =

sense strand

Glucocorticoid-Induced Glaucoma • Glaucoma TM cells express less GRβ compared to normal TM cells • Glaucoma TM cells are more _____ to GCs compared to normal TM cells

sensitive

The metaphase spindle - The cohesin complex is cleaved by _______.

separase

U2 snRNA recognizes the

sequence flanking branch point A

M-Cdk or Cyclin B/Cdk1 phosphorylates specific ____ residues in lamins.

serine

CDKs are ___________________ ; require cyclin for activity.

serine/threonine protein kinases

• The tail -- CTD (carboxy-terminal domain) - is a _____________ repeated ~50 times. CTD is phosphorylated on serine or threonine. • All of the TFII complexes except for TFIIH are left behind as RNA pol II moves forward.

seven amino acid sequence (Tyr.Ser.Pro.Thr.Ser.Pro.Ser)

1. lac operon = (e.g., lacZ encodes β-galactosidase)

several genes encoding enzymes to help degrade lactose

Apoptosis is induced by p53 when

severe DNA damage is detected and cannot be repaired.

Eating mushrooms that produce alpha-amanitin causes

severe gastrointestinal discomfort and liver damage

Effector caspases contain a

short pro-domain

miRNAs in Cardiac Ischemia - affecting the regulation of number of proteins being expressed. - miRNA's can be increased which means _________.

silencing of the protein

pre-mRNA Processed by Splicing (Removal) of Introns - The primary RNA transcript must be further processed by removing introns - Splicing must be accurate to within a ______ base pair since a mistake would throw the whole coding sequence out of register and scramble the protein sequences

single

Classification of Viruses - Class IV:

single stranded RNA (+ strand; prokaryotes Qβ, human HCV & SARS-CoV-2)

Classification of Viruses - Class V:

single stranded RNA (- strand; human Influenza virus)

Classification of Viruses - Class II:

single stranded, positive strand DNA (Only prokaryotic M13, φ174)

HIV has two __________ surrounded by a double-protein shell and an outer envelope layer.

single-stranded RNA (+ssRNA)

Condensins then replace cohesins except where

sister chromatids are held together at the centromere

Post-transcriptional Regulation of Gene Expression by miRNAs & siRNAs • Perfect match of siRNA strand in RISC leads to ___________?

site specific cleavage of target mRNA (catalytic)

Defects in mRNA Transport Cause Diseases - A human disease that results from aberrant nuclear retention of a particular mRNA in the severe muscle wasting disorder, myotonic dystrophy (DM) type I - Patients suffering from this disorder experience

skeletal muscle myotonia and degeneration, which ultimately can be fatal

RNA tumor viruses: ___ transformation by retroviruses

slow

Introns are removed from pre-mRNA by splicing carried out by spliceosome complex - Each small nuclear RNA (snRNA) plus its protein partners forms a ________ or "SNURP" • SNURPS assemble at the splice sites at the intron/exon boundaries.

small nuclear ribonucleoprotein (snRNP)

Contain dsDNA: Simian virus 40 (SV40)

small spherical virus that causes cancer in monkeys by inserting its DNA into the host chromosome.

5' Splice Sites, 3' Splice Sites, and Branch Sites on the pre-mRNA are Recognized by ____ of SNURPS

snRNAs

• The folded conformation of a protein is determined

solely by its amino acid sequence

Retinoblastoma (Rb) - In sporadic cases both normal alleles must undergo

somatic mutation

Eukaryotes use many more gene-regulatory proteins (activators, repressors, silencers, insulators, co-activators) --->

some are DNA-bound 100's to 1000's of bases away from (+1)!

Cytoplasmic pathway -

some proteins remain in the cytosol or are transported to other organelles

In eukaryotes, Met instead of fMet is used but a ____ _____ ____ is used.

specialized initiating tRNA

Translation starts at

specific initiation sites

zif268 (3 zinc fingers) bound to DNA this is a

specific transcription factor

Initiation of transcription: Upstream elements close to promoter bind

specific transcription factors

What shape is the influenza virus?

spherical

Chromosome Segregation - Sister chromatids separate and move towards the

spindle poles

The splicing machinery in eukaryotes is known as the

spliceosome

Eukaryotic Introns are removed from pre-mRNA by splicing carried out by

spliceosome complex

Pre-mRNA Splicing and Pathogenesis of Human Diseases - A probabilistic analysis indicates that over 60% of human disease-causing mutations affect _____ rather than directly affecting protein coding sequences. - Exon sequencing has been used to identify disease specific mutations but is only successful some of the time

splicing

(+) ssRNA animal virus replication (e.g., HCV) - Step 1:

ssRNA serves as mRNA • (+) ssRNA --> polyprotein --> viral proteins

Classification of Viruses - Class VI:

ssRNA viruses reverse transcribed to a dsDNA intermediate (retroviruses / lentiviruses like HIV)

Poly(A) tail is required for ______ of the mRNA and for its translation by the ribosome

stabilization

Protein folding assisted by chaperones. - They bind to and _______ unfolded or partially folded polypeptides.

stabilize

Introduction to protein synthesis - rRNA =

stage

Genetic code - 61 codons code for amino acids - 1 codon (AUG) is the ____ _____

start codon

Checkpoints are activated not only during normal cell cycle progression but also in response to internal and external _____ signals.

stress

Cancer cells have an abnormal ability to survive

stress and DNA damage

Prokaryotic If -10 box is exactly TATAAT, it is considered

strongest sigma binder

Some of these antibiotics exploit _____ _____ between bacterial and eukaryotic ribosomes.

structural differences

Cistron =

structural gene

What is the function of glycosylation?

structural or informational (protein folding, activity, stability, targeting)

Transport of unfolded proteins to

subcellular organelles

Mitochondrial chaperones facilitate transport and

subsequent folding of the polypeptide chain within the organelle

Viruses vs Cells: - Viruses do not contain ribosomes but ______ host cell ribosomes to make proteins.

subvert

It is the mediator complex that integrates all inputs and directly transmits "______" information to RNA pol II to push on or pause

summary

p53 is a tumor ______ gene product

suppressor

In the HIV genome, env =

surface and transmembrane proteins

p53 can also repress expression of genes that promote cell ____ (e.g., cyclins, Bcl-2)

survival

Inert virus particle (virion)

surviving outside the host cell

RNA polymerase II for class II genes (protein-coding genes) to

synthesize mRNA

Retrovirus replication: Virion-held reverse transcriptase (RNA-dependent DNA polymerase)

synthesizes DNA from a genomic ssRNA template

Appreciate that, in prokaryotes, a single RNA polymerase transcribes

t-RNA, r-RNA, and m-RNA coding genes

Decoding mRNA by transfer RNA (tRNA) - mRNA codons, a set of three-nucleotides specify a particular amino acid. - _______ decodes mRNA. One part of tRNA binds to an amino acid and another part recognizes the nucleotide sequence that encodes that amino acid in an mRNA.

tRNA

Initiation in Eukaryotes Step 1: - (1) elF1A prevents

tRNA binding to the A site.

RNA pol III transcribes what?

tRNA in conjunction with general transcription factors

RNA polymerase III for class III gene

tRNA, 5S rRNA, and small RNA genes

What are the several minor genes in the HIV genome?

tat, rev, nef, vif, vpr, and vpu

The HIV genome has a regulatory ____ and ____ genes are each encoded as two segments that are spliced together at the RNA level during gene expression.

tat;rev

Human T-cell Leukemia Virus - viral genome contains ______ which stimulates viral transcription

tax gene

Introduction to protein synthesis - mRNA =

template

The two DNA strands are first separated locally. Then, antisense DNA strand serves as a _____ for building new RNA

template

G0 phase

terminally differentiated cells withdraw from cell cycle indefinitely

Puromycin - An antibiotic that binds to the A site and and _________________.

terminates polypeptide synthesis

Genetic code - 61 codons code for amino acids - 1 codon (AUG) is the start codon - 3 codons (UAA, UAG, UGA) are ______ _______.

termination codons

This tail also represents process of

termination of DNA transcriptionby RNA polymerase machinery

Rho (ρ)-independent termination of transcription in prokaryotes • End of a gene marked by ___ ____ that forms an RNA hairpin. • When RNA polymerase sees hairpin structure in growing RNA, followed by a poly-(U) stretch, it terminates synthesis because U-to-A hydrogen bonding between RNA and template DNA is weak:

terminator sequence

Where are the amino acids added during elongation?

the C-terminal end of a growing polypeptide chain

When a normal cell and a cancer cell were fused, the resulting hybrids lost the ability to form tumors in mice. This suggests that

the cancer cell alleles are recessive to the normal cell alleles

Once post transcriptional processing is complete, (ie. Capping, Polyadenylation, Exon splicing, intron removal and methylation),

the mRNA message remains associated with hnRNP proteins in a messenger ribonuclear protein complex, mRNP

Future Therapeutic Approaches •Personalized and precision medicine • What is precision medicine? •How will this be accomplished? •What are the hopes? •What are the ethical dilemmas?

the medicine is precisely attacking the cause of that disease

Transposons for gene therapy - Gene therapy:

the process of making a gene product in a cell that usually doesn't make the product

Targeting proteins to endoplasmic reticulum - Step 1:

the single sequence emerges from the ribosome

Prokaryotic DNA-dependent RNA polymerase (α2ββ'ω) creates the ____ different kinds of RNA.

three

In bacteria, transcription and translation are

tightly coupled

Apoptosis plays a crticial role in

tissue homeostatis

Dicer independent:

tncRNA, smRNA, snoRNA, piRNA

RNA polymerase III for class III genes (tRNA genes, 5S rRNA genes, and small RNA genes)

to synthesize tRNA, 5S rRNA and small nuclear RNAs

Defects in mRNA Transport Cause Diseases Myotonic dystrophy (MD) type I:

toxic RNA causes nuclear retention and disease

Fibrotic Diseases Often Have Increased Expression of the EDA-Fibronectin Isoform • Glaucoma (POAG) is associated with elevated intraocular pressure (IOP) • Increased fibrosis within the _____ ______ (TM) causes this elevated IOP • Increased FN expression, including EDA-FN, occurs in the TM of glaucoma eyes

trabecular meshwork

In the HIV genome, tat =

transactivator of [viral] transcription

p53 is a

transcription factor

What are needed for correct functioning of eukaryotic RNA polymerases?

transcription factors

• TBP forms part of complex known as TFIID needed to recognize promoters specific for RNA polymerase II. • Binding of TFIID to TATA box via TBP is the first step of ______ ____. • Other factors then assemble... • Then RNA polymerase II itself arrives, accompanied by TFIIF (not visible), which likely helps RNA pol II bind.

transcription initiation

Regulation of DNA damage-induced apoptosis by tumor suppressor protein p53 - Several proapoptotic Bcl-2 family proteins are ___ ____ of p53, such as Bax, Noxa, PUMA

transcriptional targets

Acetylation

transfer of an acetyl group

Tumor viruses can cause cancer: - Some animal viruses can permanently alter the host genome -

transformation of cells

Post-transcriptional Regulation of Gene Expression by miRNAs & siRNAs • Significant but incomplete base-paring by miRNA leads to

translational arrest

Further methyl groups may be added to ribose sugars of first one or two ribonucleotides of growing mRNA (in some higher eukaryotes), giving "cap1" or "cap2". cap2 typical of mammalian mRNAs ---> appears to increase

translational efficiency at next stage of function

Puromycin - An antibiotic that binds to the A site and and terminates polypeptide synthesis. - The structure is similar to the 3'-end of an aminoacyl-tRNA. - Peptidyl puromycin cannot ___________.

translocate to the P site

Eukaryotic gene expression can be controlled at any of six different stages including the

transport of mature RNA from nucleus to cytoplasm

Basic structure of DNA transposon • Inverted repeats at either end. Often labeled as insertion elements or insertion sequences (IS); the exact sequence of the IS and the configuration is one of the ways that we classify transposons. • Gene that codes a _________ • Transposons can also carry other genes within the transposable element such as genes of antibiotic resistance • The host also has sequences that are target sequences. These specific sequences are repeated on either side of the transposon.

transposase

Bacterial transposon - In bacteria, the insertion element transposon is a very simple transposon. - It encodes a ______________________.

transposase and inverted repeat elements

Future Therapeutic Approaches •Personalized and precision medicine •What is personalized medicine? •How will this be accomplished? •What are the hopes? •What are the ethical dilemmas?

treat each patient differently and the therapy is geared for each of these patients to respond

Growth factors bind to the receptor and

trigger the signaling pathway

A codon is a

triplet of nucleotides that code for a specific amino acid

Increased Cell Division + Normal Apoptosis =

tumor

Normal Cell Division + Decreased Apoptosis =

tumor

Structural features of tRNA - In 3D, a tRNA has the form of a _______. - This ___ shape is required for tRNA to fit into ribosomes. - Different tRNAs share similar overall structures but they also possess unique identifying sequences that allow the correct amino acid to be attached and aligned with appropriate codon in mRNA.

twisted L.

How many pathways are involved in protein sorting?

two

There are ___ tRNAs for methionine.

two

RNA polymerase II transcribes eukaryotic genes that produce mRNA and code for proteins • Since many protein-encoding genes vary markedly in expression depending on cell type, a variety of specific transcription factors are needed for expression of certain genes under particular circumstances: - e.g., Fetal hemoglobin is different from the adult version because

two different Hb genes expressed at two different development stages

Eukaryotic RNA Polymerase III transcribes genes from a Class III gene promoter - TFIIIC =

two subunits τA and τB, binds to A and B blocks

Chromosomal translocation - The chimeric protein has intrinsic ____ ____ activity.

tyrosine kinase

What is that major pathway of selective protein degradation?

ubiquitin-proteasome pathway

Chromosome Segregation - Active APC-Cdc20 complex leads to ______ and degradation of securin.

ubiquitination

The Ribosome was first detected by

ultacentrifugation (designated by their rates of sedimentation)

Termination in Prokaryotes • Termination is triggered by the stop codons UAA, UAG and UGA at the A site. • The release factors (RFs) bind to the termination codon at the A site. • Release factors hydrolyze the terminal peptidyl-tRNA bond. • The complete peptide is released from the ribosome. • The uncharged ___, ____, and ___ leave the ribosome. • The ribosome recycling factor (RRF) and the hydrolysis of EF-G bound GTP aid dissociation of ribosome subunits.

uncharged tRNA; mRNA; RFs

Chaperones stabilize ______ or ______ unfolded polypeptides during their transport to subcellular organelles.

unfolded;partially

The degeneracy of the code is not _______.

uniform

Rho (ρ)-dependent termination of transcription in prokaryotes • Rho helicase (ρ) accompanies RNA polymerase along growing mRNA • Once polymerase transcribes a Rho recognition site into RNA, Rho binds this RNA site, and causes conformation change in RNA polymerase, to pause at termination site • Rho then • mRNA and polymerase fall off DNA and Rho detaches from mRNA

untwists newly-formed mRNA strand away from the DNA

Two Trans-esterification Reactions Take Place During Splicing of Two Exons • First transesterification reaction (_________)- lariat formation

unusual 2'-5' phosphodiester - bond

Protein degradation is critical to prevent build up of abnormal or ______ proteins.

unwanted

Initiation of transcription: Upstream elements facilitate initiation. • _____ elements increase the efficiency of RNA pol II binding and the efficiency of initiation of transcription (formation of 1st 3'-5' phosphodiester bond of the growing RNA strand) • ______ elements are bound by specific transcription factors

upstream

Enhancer DNA sequences can be the same types of recognition sequences as in ________ (e.g., Octamer and AP1 elements)

upstream control elements

Initiation of transcription: Upstream elements facilitate initiation. • RNA polymerase II can bind and initiate transcription at a minimal promoter consisting of just initiator and TATA boxes. However, this is extremely inefficient unless

upstream elements are also present.

Two Trans-esterification Reactions Take Place During Splicing of Two Exons • Second transesterification reaction (________)- lariat excision

usual 3-5' phosdiester bond

Human Papilloma Virus (HPV) - results in uncontrolled cell growth and

viral replication

Gene Therapy - In vivo

viral vector or non-vector directly given to the individual and this modifies gene expression within the target cells.

In the HIV genome, nef, vif, vpr, vpu =

virus maturation, host cell modulation

• The message contained within the mRNP must be transported out of the nucleus and into the cytoplasm before it can be utilized by ribosomes to make protein • The nucleus is separated from the cytoplasm by two membranes which form the nuclear envelope • Like the plasma membrane, the nuclear envelope membrane forms a ___________ phospholipid bilayer with numerous associated proteins

water-impermeable

Prokaryotic If -10 box is TACAAT, it will be a ____________ than TATAAT.

weaker sigma binder

When does the lifecycle of virus start?

when the viral DNA or RNA enters the host cell

Where are viral particles assembled?

where the genome replication occurs

whereas white blood cells make antibodies.

α1-antitrypsin deficiency is a genetic disorder caused by mutations that affect folding of ____ which aggregates and accumulates in the ER.

α1-antitrypsin

When glucose runs out, other sugars must be consumed --> Genes (and encoded proteins) for using these other sugars (such as lac operon for lactose ) need to be turned on:... like

β-galactosidase

What is a LINE?

• 17% of human genome (but only ~100 are active) • Long interspersed nuclear elements (~7000bp) that are non-LTR retrotransposons • mRNA translated into reverse transcriptase

What is the structure of a non-LTR retrotransposon?

• 5'UTR- DNA polymerase II promoter • ORF1 - Endonuclease • ORF 2 - Reverse transcriptase and RNAse H • 3'UTR - Poly A

The Ribosome • Large complexes of discrete rRNA molecules and proteins

• 65% rRNA • 35% ribosomal proteins

Post-transcriptional Regulation of Gene Expression •Alternative mRNA splicing •DNA methylation (CpG islands) •Histone acetylation & methylation •Non-coding RNAs:

• 98% of transcriptional output in humans! • Dicer dependent: miRNA and siRNA • Dicer independent: tncRNA, smRNA, snoRNA, piRNA

DNA Transposons

• A DNA transposable element (TE) that can change position within a DNA sequence • Sometimes called a "jumping gene" • It needs a transposase enzyme

Retrotransposons

• A type of transposon that must go through an RNA intermediary for transposition • Requires an integrase and reverse transcriptase

Mechanisms of Gene Delivery - Viral Delivery

• AAVs • HSV • Lentivirus • Other retroviruses

Gene Therapy - Viral Vectors - Types of viral vectors

• Adeno Associated Viruses (AAVs) • Lentiviruses

•Increased expression of GRβ can cause resistance to GC therapy

• Asthma • IBD • Rheumatoid arthritis • COPD

Characteristics of apoptosis - Morphological features:

• Chromatin condensation • Cytoplasmic blebbing • Apoptotic bodies • Cell shrinkage

What are classes of transposons?

• DNA Transposons • LTR Retrotransposons • Non-LRT Retrotransposons • Autonomous • Non-autonomous

Defects in mRNA Transport Cause Diseases • Spinal muscular atrophy (SMA)

• Decreased levels of SMN (survival motor neuron) proteins • SMN essential for muscle development and movement • Aberrant RNP processing of mRNA prevents export from nucleus

Half-lives of eukaryotic proteins vary considerably from 30 sec to several days.

• Defective proteins are short-lived • Many regulatory molecules are short-lived • Some proteins are long-lived (e.g., hemoglobin)

Gene Therapy - Research Applications

• Discover pathogenic pathways • Generate disease models • Therapeutic target validation • Preclinical testing of gene therapy

The Ubiquitin-proteasome pathway includes three separate enzymes:

• E1 activating enzymes • E2 conjugating enzymes • E3 ligases

Gene therapy needs •Chromosome targets

• Exons • Introns • Noncoding DNA

Alternative Splicing of the Glucocorticoid Receptor •GCs unsurpassed as anti-inflammatories •Alternative splicing to form:

• GRα = ligand activated transcription factor • GRβ = dominant negative regulator of GC activities

Myocilin Glaucoma • Novel mechanism of action

• Gain-of-function • Induces protein stress

Gene Therapy - Technology - CRISPR/Cas9 genome editing

• Gene knockout • Directly editing mutation

Gene Therapy - Viral Vectors - Advantages

• Generally good transduction efficiency • Controlled tissue tropism

Clinical In Vivo Gene Therapy - Leber Cogenital Amaurosis (LCA) • Blinding eye disease in young children • Multiple genes involved (>26)

• Genotype patients & identify individuals for gene therapies • Subretinal injection of viral vector • Recovery of vision Examples • RPE65 • GUCY2D

Genetic alterations leading to cancer

• Growth-promoting proto-oncogenes • Growth-inhibiting tumor suppressor genes • Genes that regulate programmed cell death • Genes involved in DNA repair

Future Therapeutic Approaches • Treatment of mitochondrial diseases like Alzheimer's Disease, Muscular dystrophy, Diabetes, cancer, MELAS, Leigh syndrome, etc.

• How will mitochondrial diseases be treated? • What technology will be used? • What are potential problems and issues?

Clinical Ex Vivo Example - iPSC Therapy

• Identify patient's specific disease mutation • Remove skin sample from patient • Isolate fibroblasts • De-differentiate to generate iPSCs & correct gene defect (casper-9) • Differentiate to specific cell type to deliver back to patient

DNA transposons - The insertion site is replicated, not destroyed when a transposon inserts. - This means that the insertion site is still a good place for future transposon insertions - Sometimes transposons insert close to one another because of these insertion sites - When this happens, you can get longer and more complex transposons

• If there are no mutations, you can get both independent and complex transposition • If mutations occur, it is possible to have a complex transposon • In a complex transposon, it is possible to move host DNA along with the transposon.

Future Therapeutic Approaches

• In utero diagnosis and treatment of genetic diseases • How will genetics of fetuses be identified? • How will disease genes be modified in utero? • That are the ethical concerns?

Conversion of proto-oncogenes to oncogenes - Chromosomal translocation:

• Inappropriate expression of the gene due to translocation of growth regulatory gene under the control of different promoter • Fusion of two genes produce a hybrid gene encoding a chimeric protein

Role of TGFβ2 in Glaucoma • Increased TGFβ2 expression in glaucoma • TGFβ2 drives fibrosis by: • Target validation and development of mouse model

• Increased ECM synthesis • Decreased ECM degradation • Covalently cross-linking ECM

Gene Therapy - Non-viral Vectors • Advantages

• Increased cargo capacity • Little inflammation or IR

Myocilin Glaucoma • Not loss-of-function • Ad5.hMYOC mutant delivery to mouse eyes:

• Induces ocular hypertension • Genotype/phenotype correlation

Gene Therapy - Viral Vectors - Disadvantages

• Inflammatory & immune response • Insertional mutagenesis • Cargo limits

Frameshift Mutation

• Insertion or deletion of nucleotides within coding region of a gene (not a multiple of 3) so that the reading frame is altered. • All the amino acids after deletion and insertion will be different.

MicroRNA (miRNA) • Small double stranded RNAs transcribed from genomic DNA

• Intronic regions of protein coding genes • Exonic or intronic regions of non-coding RNAs • Individual transcription units under control of separate promoter

Mechanisms of Gene Delivery - non-viral delivery

• Liposomes • Polymers • Nanoparticles • "Gene gun" • Electroporation

Gene Therapy - Non-viral Vectors • Types

• Liposomes • Polymers• Nanoparticles • "Gene gun" • Electroporation

What is the structure of LTR retrotransposon?

• Long terminal repeat (LTR) retrotransposons • LTR sequence at each end (5' and 3') • Some able to make viral-like particles • Reverse transcriptase • RNASE H • Integrase • Needs a host TSR (target site recognition repeat region) to incorporate

Gene therapy needs • Genetic diseases:

• Loss of function mutations • Gain of function mutations

LINEs

• Make up 17% of the genome • 99.9% of these sequences are no longer capable of retrotransposition (called dead or inactive) • BUT 0.1% are capable and may jump (and may cause disease)

Gene Duplications - Creation of pseudogenes • DNA slippage as replication occurs: similar repeated sequences within the genome can result in DNA replication slippage

• Microsatellite and LINE and SINE sequences are some of the repeated sequences where this can occur • When this type of duplication occurs, there tends to be partial copies of a gene that includes the introns

Almost all transposons in human genome are silent. • Only ~100 retrotransposons are active in human genome • Transposition inhibited by:

• Mutations within transposons • Active silencing by siRNAs and chromatin remodeling

miRNAs

• Non-perfect base paring (e.g. 6/8 nts) with target mRNA • Single miRNA can target 100s of mRNAs • Single mRNA can be targeted by multiple miRNAs • Silencing via translational repression

Knudson's "two-hit hypothesis" of oncogenesis:

• One genetic change ("first hit") is inherited • Spontaneous mutation ("second hit") in one of the retinal cells which already carry the first mutation

Gene therapy needs •Alterations in signaling pathways

• Pathogenic pathways • Cytoprotection/neuroprotection

siRNAs

• Perfect base pairing with target mRNA • Silencing of specific mRNA (only one target) • Silencing via mRNA cleavage and degradation

Clinical Gene Therapy

• Preclinical and clinical efficacy & safety must be demonstrated prior to approval • Vectors - DNA and RNA • Somatic and mitochondrial DNA • Ex vivo gene therapy •In vivo gene therapy: mRNA covid vaccine

miRNAs in Disease: Biomarkers

• Profile miRNA expression patterns (biomarkers) • Diagnose type & state of tumor • miRNAs in blood after cardiac muscle damage • miRNAs in exosomes

The Ribosome • Composed of two unequal subunits:

• Prokaryotic ribosome (70S) - 30S and 50S • Eukaryotic ribosome (80S) - 40S and 60S

Roles of miRNA In Development • Originally identified in plants and C. elegans • RNAi discovery awarded Nobel Prize (Fire and Mello in 2006)

• Regulates development, cell proliferation, cell fate determination, and apoptotic pathways • Temporal and tissue specific miRNA expression patterns

Gene Therapy - Non-viral Vectors • Disadvantages

• Selective targeting?• Duration of delivery?

Gene Duplications - Creation of pseudogenes • Retrotransposition

• These duplicated genes tend to have no introns as they are made from mRNA • They also tend to lack a promoter as this is not generally transcribed in the mRNA

Transposons do not exist outside a host DNA

• They have no origin of replication • They often lack the enzymes necessary for independent transposition • You can't "catch" a transposon like you would a virus or a bacteria

• Transposons are not just "junk" DNA

• They influence recombination rates • They influence mutation rates • They influence gene regulation

In humans, transposons elements are generally inactive. Transposons can move via two different mechanisms. • Replicative transposition - ???

• This does increase the number of transposons in the DNA • Involves single stranded cuts

Transposons can drive genetic diversity (genome evolution)

• Translocation of genome sequences to make new genes • Shuffling of exons • Generate new regulatory regions • Repair of double-stranded DNA breaks

replicative transposition • Structure of a replicative transposon is more complex

• Transposable element is duplicated so that it does not leave the host DNA strand • It requires an additional enzyme (resolvase) • It also requires an internal resolution site • These are often called complex transposons

conservative transposition • Transposase attaches to the inverted repeats of the transposon • Creates a double stranded break (where the transposon used to be)

• Transposon then forms a hairpin and is bound to transposase • Transposase finds a suitable DNA insertion site • Transposase cuts the recipient strand with a 3 prime overhang. • Inserts the transposon into the cut DNA • Host factors then fill in the missing base pairs • Ligase seals the DNA nicks. • The host DNA attachment site is now doubled

What are the two major pathways of protein degradation?

• Ubiquitin-proteasome pathway • Lysosomal pathway

Gene Therapy - Technology - Delivery Vectors:

• Viral • Non-viral

Gene Therapy - Technology - Tissue specificity

• Viral tissue tropism • Tissue specific promoter

Experimental Uses of RNA Interference • Use siRNA or shRNA to selectively knockdown gene expression in cells/tissues •Examples:

• siRNA to knockdown Smad3 in human optic nerve head cells demonstrating the role of Smad signaling in TGFb2 fibrosis • Ad5.ID1.shRNA to identify downstream BMP signaling to inhibit TGFb2-induced ocular hypertension in mice

Codon-anticodon interactions

• tRNAs base pair with mRNA codons by means of a three-base sequence on the tRNA called the anticodon. • The codon-anticodon pairing is antiparallel. • The binding of the anticodon of the tRNA to the codon in the mRNA is by complementary base pairing.

What is an ALU?

• ~11% of human genome (~1 million copies) • Specific SINE (short interspersed nuclear element) [cleaved by Alu restriction endonuclease] • ~ 300 bp repeats • Insertional mutagenesis (disease causing)

MicroRNA (miRNA) • Post-transcriptional silencing of mRNA/protein expression

•Binds to mRNA (usually 3'-UTR) •Translational repression (miRNA) •mRNA cleavage and degradation (siRNA)

Therapeutic Potential of RNAi • Inhibition of pathogenic pathways • Problems delivering siRNA ?? • Approaches using shRNA vectors (plasmids & viruses)

•Ribonucleases •Innate immune response •Delivery of siRNA inside cells •Targeting specificity

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