Exam 1!

over 25%

% of bases posttranscriptionally modified in tRNA

3 ribosomal sites

(E) exit site, (P) peptidyl transfer site, (A) incoming aminoacyl tRNA site EPA environmental protection agency

what tRNA's are bound in the pretranslocational state

A and P site

How does altered Poly A site affect splicing and regulaton

Altered poly A tail choice results in cancer with mRNA that have shorter UTR. In cancer cells, use of upstream poly A sites results in shorter UTR at 3' end and in some cases, escape normal regulation by mRNAs.

How is alternative splicing gets regulated?

Alternative splicing is regulated by short regulatory sequences on the RNA: 1. Splicing enhancers: intronic or exonic enhancer -->short RNA that bind to proteins and enhance the formation of functional spliceosome 2. Splicing silencer: intronic or exonic silencer. -->Short RNA that binds to proteins that silence the formation of functional spliceosome. .

Helix-Turn-Helix Binding Domain

Most common domain in prokaryotic TFs 2 alpha helices with turn in between One helix is recognition fitting into major groove, other helix stabilizes binding by interacting with DNA backbone HTH symmetric dimers can recognize sequences close together Homeodomain subgroup: HTH + 3rd helix for additional stabilization Ex. Hox genes organizing human body plan in early development

Chromatin Immunoprecipitation (ChIP)

Determining DNA sequences bound by TFs *in vivo* (living cells) Results typically correspond to those in SELEX experiments--shows that high affinity between DNA and DNA-binding proteins can accurately predict TF-DNA interaction in a living cell even w/ the more complex DNA/protein editing/modifying processes occuring in cells

Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

Determining binding specificity *in vitro* (tube) Start w/ solution of TF protein of unknown DNA-binding specificity Add large pool of short dsDNA fragments with randomly generated 12-nucleotide middle regions Remove all non-bound DNA via gel electrophoresis (i.e. gel-mobility shift assay) Remove bound proteins from remaining fragments, amplify fragments w/ PCR Repeat process multiple times to isolate exact protein-bound sequence

What is a good probe design?

-25-mer -Unique -Sensitive (low complexity probes have low specificity = lots of false positives) -Not able to hybridise itself (Self hybridised probs are unavailable and decrease value) -Within a certain thermodynamic range. Probe target must have similar melting tempratures. (Too high = difficult to bind, decreased value. Too low = Binds too easily, elevated value. The 'melting' temperature is sequence dependant).

What is the function of the cap binding complex and its relation with mRNA cap?

-5' cap recognizes the CBC binds to it. -the CBC is important in subsequent maturation steps: - 1) Splicing 2) 3' processing (poly A tail) - the CBC stabilizes the 5' end of the RNA against exonuclease..

Can a MONOMERIC PROTEIN with a SINGLE LIGAND BINDING site exhibit cooperative behavior?

-A MONOMERIC PROTEIN with a single ligand binding site can NOT EXHIBIT COOPERATIVE BEHAVIOR.

What is gene expression?

-A highly-regulated process that determines whether a gene is switched on or not

describe the translocation step of elongation

-A+P move to P+E sites -mRNA moves along by one codon -EF-G (eEF2) and GTP bins and moves ribosome by one position -not charged tRNA in E site ejected -GTP hydrolysis and EF dissociates

2 common motifs

-helix-turn-helix -zinc finger

what is the RNA world hypothesis

-idea that RNA came first before DNA -solves the issue of what came first nucelic acid or proteins -RNA stores genetic info and has catalytic activity -at some point RNA came together in a way to form a protein -DNA is more stable and modified RNA- logical RNA came first

How does antisense RNA interact with the UTR's to affect translation

-linear- on -antisense RNA- expressed elsewhere -complimentary binds SD and start of coding so R can't associated

How does temperature and the UTRs interact?

-structure has SD and AUG- R can't bind, -increase temperature melts structure -so R can bind- RNA 'thermosensors' -eg listeria

What is the eIF4E

-the eIF4E replaces the CBC in the cytoplasm and binds to the 5' cap. - 5' cap is an important determinant of translation in the ribosome b/c it binds to elF4E -also a key step in mRNA turnover: when the eIF4E dissociates from the 5', the 5' is exposed to exonuclease and get degraded

What are the main steps of processing of primary RNA transcript?

1.5'end of mRNA is 'capped' with methylated GTP 2.3' end is polyadenylated. A tail of several hundred adenosines (A) are attached 3.The mRNA is 'spliced' to remove intron regions

1 error every

10,000 added nucleotides

Ubiquitin

a 76 amino acid protein that is used to mark proteins for degradation. The COOH end of ubiquitin is attached to the -NH2 at the end of the R-group of lysine.

Transcription is conducted by

a DNA-dependent RNA polymerase II on protein-coding genes resulting into an mRNA

operon

a group of genes that are all transcribed by a single messenger RNA, and then translated separately.

selenocysteine insertion sequence (SECIS)

a hairpin loop in mRNA that binds a translation elongation factor that causes Sec-tRNA to be inserted at every UGA in mRNA

streptomycin

a miscoding agent that results in non functional proteins resistant bacteria have restrictive protein synthesis that is slower and more accuracte

eIF-4e translational control

eIF-4e recognizes the 5' cap of mRNAs-- helps to facilitate initiation of translation 4E-BP (eIF-4e binding protein) binds to eIF-4e and blocks its interaction with the eIF-4f complex cell stress stimulates the interaction of 4E-BP with eIF4-e favorable cell growth conditions lead to phosphorylation of 4E-BP which blocks its binding to eIF-4e

eIF1 and eIF3

eIF1 and eIF3 bind to the 40s subunit of the ribosome to prevent its interaction with the 60s subunit

what are the release factors in eukaryotes

eRF1 does all stop codons

codon

each amino acid is coded for by a group of 3 nucleotides

what are polyribosomes

each mRNA translated by many ribosomes simultaneously make lots of one protein at once

editing by aminoacyl-tRNA synthetases

editing the amino acid is based on shape via size exclusion; non-cognate does not fit in active site or editing site and is sterically excluding and only non-cognate fits in editing site and will be removed via hydrolysis balanced pools of amino acids minimize mismatches

ET-T and ET-G

elongation factors

greatly increase the levels of transcription, work in both orientations in DNA, and are often tissue specific

enhancers

regions of tightly packed TFs and can be located a long distance from the promoter

enhancers

How can specific TFs be found? (low abundance problem)

enrichment based on protein properties or based on affinity to DNA

describe adapter I of tRNA synthesis

enzyme pocket fit a specific amino acid sidechain residue 2 active sites synthesise site, if wrong amino acid move to editing site and is removed

Mechanisms used to regulate TF activity are similar to those for

enzyme regulation

IIeRS

example of aminoacyl-tRNA synthetase that subjects aminoacyl-adenylates to proofreading step

contain the coding sequence for the mRNA which is transcribed from its promoter

exons

NH2 of second amino acid

f-Met is displaced from its tRNA by _____; transfer results in dipeptidyl tRNA in A site and uncharged tRNA in P site

Elongation summary

for each amino acid residue correctly added to the growing peptide, two GTPs are hydrolyzed to GDP and Pi as the ribosome moves from codon to codon along the mRNA toward the 3' end 1. decoding 2. accomodation 3. hybrid state 4. translocation

Elongation

formation of polypeptide chain-- decoding mRNA, peptide bond formation and translocation GTP supplies energy for this process

eIF4

forms the pre-initiation complex binds the 5' cap of mRNA and places the 40S subunit on the mRNA in the proper orientation (eIF4f and eIF4g) mRNA is then scanned for the AUG start codon (eIF4a)

formyl residue

formyl residue on bacteria protect high energy bond in leu of EF-Tu

multiple signals are integrated at the level of __

gene expression

Initiation of translation

goal is bringing together small 40s ribosomal subunit, mRNA and met~tRNA, followed by association of large 60s subunit to from the 80S ribosome

describe export ready RNA

has 5' cap, poly A tail, spliced travels from nucleus to cytoplasm through nuclear pore complex which is tightly controlled so no wrong RNA's enter ribosome

Accuracy of aminoacyl tRNA is

high

activates valine and isoleucine, hydrolyses aminoacyl-tRNA only accepting aminoacyl groups smaller than Ile (valine)

how IIeRS proofreads, action of 2 active sites

proofreading

how aminoacylation made accurate

one less methyl group, smaller

how valine differs from Ile

list the types of point mutations

silent- new codon but same amino acid missense- new codon, different AA nonsense- sense coding to stop codon, prem termination causing a truncated protein frameshift- insertion or deletion of nucleotide shifting the reading frame

how are sgRNA's used in research and medicine

single guide RNA modify by CRISPR/Cas9 fast and easy in any genome complementary bind target ds break can integrate new DNA= knock in knock out= repair by cell mechanism- in or out- less accurate by more efficient

variant arm

site of variability on tRNA

Inhibition of translation by antibiotics

some abs are inhibitors of protein synthesis in eukaryotes or prokaryotes only prokaryotic inhibitors are clinically useful

cell or tissue specific expression

specific proteins are needed in only in specific cell or tissue types. not all proteins need to be expressed in all tissues/organs

G protein activating proteins or GAPs

speeds up Conversion of GTP to GDP (going from on to off)

what are ribozymes

ssRNA with catalytic activity eg ribosome

amino acid activated to form aminoacyl-adenylate, aminoacyl-AMP reacts with tRNA to form tRNA-aa

steps in aminoaylation

Localization protein regulation of TFs

stimulation of nuclear entry, release from membrane fast but energy intensive and more errors

step 1 termination

stop codon is encountered by ribosome

how does translation termination occur

stop codons don't have matching tRNA and are recognised by protein release factors -RF structurally similar to tRNA -bind and displace tRNA in A sites -transfers peptidyl group to water on C terminal -causes termination- free polypeptide -dissociation of large, small, tRNA and RF as no more polypeptide -bacteria have ribosomal recycling factor which binds to A site

Activation of amino acid "charging tRNA"

tRNA needs to be charged with its amino acid catalyzed by amino acyl tRNA synthetase

size

tRNAs have similar structure but vary in

isoaccepting

tRNAs that carry the same aa are

ribosomal exit tunnel

target of antibiotics because it is highly conserved yet still distinct between eukaryotes and prokaryotes; block the tunnel or interfere with peptidyl transfer at the active site - has to be big enough for protein to fit - needs to be inert without charged aa - not completely open to solvent - needs to be non-specific

why gene expression needs to be tightly controlled?

temporal regulation cell or tissue specific expression expressing genes energy intensive abnormal levels of proteins are toxic

non-overlapping code

better for mutations; evolved to minimize impact of mutations; single base changes protected from detrimental effects by making hydrophobic and charged amino acids on opposite sides of table

base pairing

between stems of tRNA

what are some functions in nucleus of long non coding RNA's

bind proteins- bring together like scaffold -bring RNA and DNA together -protein modification complexes -act as transcriptional regulator and form complex -involved in splicing

co-activators

bind to activator proteins to mediate gene activation

transcriptional activators

bind to enhancers to activate gene expression

co-repressors

bind to repressor proteins to promot gene repression

transcriptional repressors

bind to silencers to repress gene expression

TFIIB

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

TBP

binds to TATA box, part of TFIID

TFIIA

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

initiation factors

block binding into the E and A tRNA sites and prevent assembly with large subunit to ensure that initiator met guided into correct place

direct regulation of TFs (4)

compartmentalization (chaperones/inhibitors), phosphorylation/dephosphorylation, acetylation, ubiquitination/proteosomal degradation

kinetic proofreading

contributed by EF-Tu during the pause to sample; limited by availability of tRNA in sampling

Quality control

if there is a misincorporation, there is a mismatch between the tRNA and mRNA in p site that results in low fidelity in the A site that will bind a release factor and result in premature termination and degradation of protein

diphtheria toxin

inactivates EF-2 (moves P to E and A to P site) which causes an inhibition of protein synthesis toxin catalyzes ADP ribosylation (covalent modification) of EF-2

TFIID

includes TBP, recognizes Pol II specific promoter

heterodimers from DNA binding domains

increase combinatorial regulation power

Many DNA regulatory elements work together to

increase probability of transcriptional initiation

erythromycin

inhibits: prokaryotes mechanism: interacts with 50S ribosomal subunit to block the exit tunnel and release of the nascent polypeptide

streptomycin

inhibits: prokaryotic mechanism: interacts with S-12 of 30S-- inhibits initiation and causes misreading of mRNA

chloramphenicol

inhibits: prokaryotic translation and eukaryotic mitochondrial translation mechanism: inhibits peptide transferase (50s)

initiator tRNA

inserts the first amino acid, which is always methionine or a derivative.

Tetracyclines

interact with 30S subunit, blocking access of the aminoacyl-tRNA to the A site, thereby inhibiting elongation

DNA-activation domain

interacts with the co-activator

DNA-repression domain

interacts with the co-repressor

eIF5

interacts with the pre-initiation complex to form the initiation complex GTP on eIF-2 is hydrolyzed to GDP and other factors are released allowing the 60s subunit to associate with the 40s subunit

what is the structure of most snoRNA's

introns degraded flower and first processed into snoRNA transcribed by RNAP II

regulatory sites often contain

inverted repeats

Electrophoretic mobility shift assay (EMSA)

isolate DNA with regulatory elements, radioactively label, incubate labelled DNA with TFS, separate on non-denaturing gel, some labelled DNA will migrate slower (greater MW)

5'terminal nucleotide, Non Watson Crick base pairs

key features of acceptor stem

5' terminal phosphate group, 7bp acceptor stem, D arm, anticodon arm, T pseudo C arm, 3'CCA sequence with 3 free OH

key features of tRNA

what is a ribosome

large ribonuclear protein containing non coding RNA and proteins 2 subunits contribute to active site which remain separate until binding of mRNA

acceptor and T pseudo C, anticodon loop and D

legs on tRNA

5bp

length of anticodon arm

3-21 nt

length of variant arm

Allosteric regulation of TFs

ligand binding, covalent modification, addition of subunits, unmasking fast but energy intensive and more errors

step 1 elongation

mRNA is bound and charged initiating methionine is in the P-site of the ribosome which is bound to the first AUG

what are some functions in cytoplasm of long non coding RNAs

mRNA stability translation circular RNA form small peptides

polysomes

many ribosomes on the same mRNA protein as it is transcribed

EF-2

moves AA and tRNA from P site to E site and from A site to P site

Coactivator CBP can integrate inputs from

multiple transcriptional activators

describe the transpeptidation step of elongation

new peptide bond forms peptidyl group in P site- aminoacyl group in A site -transferase activity- transfer existing pp to new AA -still a chance ribosome will reject a wrong tRNA after GTP reacts and preferentially dissociate

describe prokaryotic initiation

no 5' cap polycistronic ribosome binding sites upstream of start codon- shine-dalargno sequences specific met- fmet= formyl group- makes sure met is first codon and later processed away

what are transfer RNAs

non coding RNAs match nucleotide sequence to amino acid

what are small nuclear RNAs

non coding RNAs part of spliceosome

Transcriptional coactivators

non-DNA binding components that interact with multiple activators, can directly recruit GTFs or modify chromatin structure

TFIIH

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

what are long non coding RNA's

pieces of RNA longer than 200 nucleotides variety of functions involved in gene expression -transcriptional and post transcriptional regulation -cis and trans acting

wobble position

position does not make perfect base pair while first two positions do not follow WC base pairing; U can pair with A or G that otherwise couldn't occur in helix due to the physical constraints of helix; inosine can occur in the anti-codon of some tRNAs and can bp with U, C, and A

maintaining tRNA's integrity

posttranscriptionally modified bases in tRNA are not essential for

mRNA structure

prokaryotes have a shine-delgado sequence before the AUG start codon, the ribosomal binding sequence, spacing is key between SD and AUG; open reading frame because they are polycistronic, have an open triphosphate on the 5' end eukaryotes have a 5' cap, methylated guanine, on their triphosphate 5' end and a poly A tail

Four ways activators can function

promote binding of more regulators, recruiting RP to the promoter, releasing RP to begin transcription, releasing paused RP

regulation of transcription is mediated by __ and __

promoter and enhancer/repressor elemts

___ are required for transcription initiation

promoter proximal elements

these elements are sites for DNA binding TFs usually within 200 bp of TSS

promoter proximal elements

Activators and repressors control RNA polymerase function at...

promoters

Total protein regulation of TFs

protein synthesis energy efficient and accurate but slow

Control of Eukaryotic translation

protein synthesis is high energy stresses (viral infection, oxidative stress, ER stress, nutrient limitation) can lead to inhibition of translation

mRNA masking

proteins can bind to mRNA and block its ability to be translated eg: iron regulation

release factor proteins

proteins that stops translation, one is a protein similar to tRNA and the other is G protein

RNA polymerase I

rRNA

what is the active site of ribosomes

rRNA

what are ribosomal genes

rRNA transcribed from one rDNA gene repeates transcribed by RNAP1 in nucleolus 2 types of chemical modification

describe the steps of removing introns

recognition sequences = 5' and 3' splice site branch point attacks 5' splice site branch point attached to A-OH (loop) separates exon 1 and intron G-OH of exon 1 attacks G-P of exon 2- transestification 2 forms bond between exon 1 and 2 with splice junction- exit nucleus lariat forms- excised intron which is degraded

DNA-binding domain

recognize and bind to proximal-promoter sequences or enhancers/silencers

amino acyl transferase

recognizes correct amino acid with ATP-- ATP is used to activate the amino acid 1. specific tRNA is brought in and bound by AA (synthetic site) 2. Amino acid is checked (hydrolytic site of enzyme removes incorrectly bound AA) 3. AMP is released 4. Charged (has AA) tRNA is released from the enzyme

describe the different types of exons

regulated-cell type or developmentally specific constituative- same exon in all cells cassette exons- arrays of alternative exon forms giving receptor diversity

regulation of galactose metabolic genes by GAL4 transcriptional activator

Step 1: Basal TF TBP binds to the core promoter TATA box and recruits TFIID. Step 2: GAL4 gene is induced in response to galactose. Step 3: DNA-binding domain of GAL4 binds to enhancers (UAS). Step 4: DNA-activation domain of GAL4 binds to basal TF machinery to recruit RNA polymerase. Step 5: DNA-activation domain of GAL4 can also binds to mediator to recruit RNA polymerase. Step 6: Transcription of GAL genes are ON.

non-stop mediated decay

this occurs when the transcript lacks a stop codon and continues to read poly A tail resulting in lysines at the end of protein; danger signal that recruits proteins to disassemble ribosome, degrade RNA, and degrade protein

why is tRNA synthesis tightly controlled

tightly control the coupling of AA to tRNA as ribosome cant tell if tRNA has right AA

__ is obtained by unique combinations of expressed TFs, not via unique __ DNA binding proteins

tissue specificity

EF-G

translocation is mediated by ____, another GTPase

elongation - translocation

translocation of mRNA-tRNA complex requires EF-G that works via molecular mimicry of tRNA bound to EF-Tu tRNA; peptide bond formation induces a chemical change by the ribosome into a hybrid state that EF-G can bind to 1. movement of the large subunit c-terminal ends into adjacent site by brownian motion 2. conformational change in 30S subunit 3. GTP hydrolysis to move tRNA via interaction of the stalk + elbow of tRNA moving into exit site - conformational changes between the large and small subunits occur separately

G proteins

used for a variety of signal transmissions within the cell, as well as between the outside of the cell and the inside

two points of energy input into system of protein synthesis

via GTP hydrolysis to add amino acid onto tRNA and peptidyl transfer and translocation

G proteins "ON"

when a g protein binds to a molecule of GTP. The G protein slowly hydrolyzes the GTP to GDP.

what is 2'-O-methylated nucleotide

when snoRNA methylates ribose sugar of any base

acceptor stem and anticodon loop

where aminoacyl-tRNA synthetase contacts tRNA

Translation: Elongation

where the polypeptide chain is synthesized one amino acid at a time.

20-25A

width of tRNA

Most common DNA binding domains in humans

zinc fingers, homeodomains, helix-loop-helix, leucine zipper

Explain how Splicing Is Temporally and Functionally Coupled with Multiple Steps in Gene Expression

• Splicing can occur during or after transcription. • In general splicing begins as a cotranscriptional process and continues as a posttranscriptional process. • The transcription and splicing machineries are physically and functionally integrated. • Splicing is connected to mRNA export and stability control.

Describe the characteristics of splicing in pairs

• Splicing depends only on recognition of pairs of splice sites. • All 5′ splice sites are functionally equivalent, and all 3′ splice sites are functionally equivalent. • Splicing junctions are recognized only in the correct pairwise combinations.

Explain how Splice Sites Are Read in Pairs

• Splicing depends only on recognition of pairs of splice sites. • All 5′ splice sites are functionally equivalent, and all 3′ splice sites are functionally equivalent. • Splicing junctions are recognized only in the correct pairwise combinations. .

Describe the mRNA Splicing & Export are Coupled Processes

• Splicing in the nucleus can influence mRNA translation in the cytoplasm. • nonsense-mediated mRNA decay (NMD) - A pathway that degrades an mRNA that has a nonsense mutation prior to the last exon. • EJC is usually tripped off by the ribosome during the first round of translation. • A premature stop codon means the ribosome dissociates and EJC remains which then recruits additional proteins such as Upf which recruits a decapping enzyme resulting in mRNA degradation.

Leucine Zipper (bZip)

"coiled coil" of 2 alpha helices with leucine every 7 residues Helices straddle DNA w/ their basic (b) region Always form homo/heterodimers Ex. AP1: c-Fos/c-Jun heterodimer controlling cell growth by regulating cell cycle

Helix-Loop-Helix (HLH)

"zipper" form is interrupted by loop in each of the DNA-straddling helices Ex. c-Myc: if amplified, can assist in transforming cells to cancer cells

Aminoacyl-tRNA synthetases

- Activate amino acids by charging CCA ends of cognate tRNAs - Many aa-tRNA synthetases edit incorrect amino acids (aa)

Anticodon arm

- Anticodon stem - Anticodon loop End that decodes the codon Pairs with the codon on the mRNA

T arm

- Contains TΨC sequence - Signature Sequence universally present in tRNA - T = Ribothymidine; a modified thymidine present only in tRNA - Ψ = symbol for pseudouracil - C = cytidine Facilitates binding of the a.a. to the A site

activity of transcriptional regulators can be regulated through...

- covalent modification (e.g. phosphorylation, acetylation) - subcellular localization - conformational change upon binding to a small molecule (allosteric regulation)

What is the torpedo model of termination?

-- A ribonuclease binds to 5' end of cleaved RNA (i.e., to the downstream fragment) and "chases" the elongating Pol II as it moves further downstream of the PAS.Starting at cleavage site, the nuclease degrades the downstream transcript in 5'-3' fashion. When the nuclease "catches up" to Pol II then termination is triggered; (source-- Proudfoot review).

What sequence does Cleavage and Polyadenylation Specificity Factor (CSPF) recognize?

-- AAUAAA

The spliceosome assembly requires energy input from what?

-- ATP hydorlysis

Where are ribosomal RNA synthesised?

-- All but 5S are synthesized in a nuclear structure called the nucleolus by RNA Pol I.

What are the functions of modified RNA bases?

-- Alter the base pairing ability -- Alter the stability of the RNA secondary structure -- Alter the ability of the RNA to interact with proteins

What are the two main classes of snoRNA?

-- C/D box snoRNAs, which are associated with methylation -- H/ACA box snoRNAs, which are associated with pseudouridylation (chi)

How does D effect the stability of tRNA?

-- Destabilizes it

What is ISE?

-- ISE: intronic splicing enhancer

What is ISS?

-- ISS: intronic splicing silencer

Are modified bases of RNA incorporated during transcription?

-- No

Does ribosomal RNA encode proteins?

-- No

What is the splicing reaction catalyzed by?

-- RNA -- not protein

What are ribozymes?

-- Self splicing introns

What does mRNA splicing do?

-- Splicing removes introns and joins exons together so as to enable translation of the correct protein

What is similar between group II introns and spliceosomes in how they work?

-- They both use an A and stabilizing metals (2) to attack the the phosphate and join the extrons

What does Cleavage Factor II (CFII) recognize?

-- UGUA repeats

What conserved sequence elements mark the intron boundaries?

-- Y, pyrimidine; R, purine; N, any base

When do covalent modification occur to RNA?

-- after termination step

What is Frontotemporal dementia with parkinsonism (FTDP-17) is often caused by?

-- silent and intronic mutations within a chromosome 17 gene (MAPT) encoding a microtubule-associated protein (tau)

What is Cleavage and base modification of rRNA and covalent modifications of tRNA and some snRNAs is guided by?

-- small nucleolar RNAs (snoRNA)

What is an example of a Microarray analysis?

-Amplification of a cytochrome P450 (22-fold over expressing) CYP6CY3 gene is associated with resistance of neonicotinoid iinsecticides in the aphid Myzus persicae -Also showed 2-16 fold over expression of gene sequence encode cuticular proteins.

What happens as the concentration of LIGAND INCREASES?

-As the concentration of ligand INCREASES, it will begin to BIND to sites. -When it binds, it INDUCES CONFORMATIONAL CHANGE in the other protein subunit of the dimer that causes its binding site to SHIFT to an open state that has HIGH AFFINITY for ligand. -A second ligand can now bind easily to the second site.

How is microarray fabrication done?

-By 'spotting' pre-synthesized Oligonucleotides.

Types of zinc fingers

-C2H2 -TFIIIA -C2C2 -glucocorticoid receptor -Zn2C6

What is the cause of "COOPERATIVE BEHAVIOR"?

-COOPERATIVE BEHAVIOR is caused by allosteric changes in associated proteins.

What is "COOPERATIVITY"

-COOPERATIVITY can be defined as a phenomenon that occurs when binding of one ligand molecule PROMOTES BINDING of additional ligand molecules.

Homeobox Protein

-CTD is DNA-Binding Domain (H-T-H) -NTD is Activation Domain -helix 3 is recognition helix, interacts w/DNA -binds to ATTA sequence in promoters

Where does alternative splicing of mRNA occur?

-In the CACL gene in Thyroid and NEuronal cells

What happens at LOW concentrations of LIGAND?

-LITTLE binding occurs.

Example of a DIMER WITH TWO LIGAND BINDING SITES:

-The example of a dimer with two ligand binding sites is the most simple example. -There are examples of cooperative transitions that involve binding of 4,6 and 8 ligands. -The more INDEPENDENT binding events involved, the steeper the cooperative transtions.

What are some promoter elements?

-The promoter consists of the core promoter which is the minimal region that can direct the initiation of transcription (TATA box) and upstream promoter elements.

What are the upstream promoter elements?

-They are also known as responsive elements and are located at 100-250bps upstream -They are short stretches of DNA that form binding sites for various transcription factors. -Each specific transcription factor binds to a specific promoter. -The number identity and location of upstream PE's vary between genes allowing complex regulation of gene transcription.

What are the bound transcription factors?

-They may interact with proteins necessary for transcription (e.g. RNA polymerase II) to increase binding affinity to that region or increase activity. 1.Some repressor proteins may bind to inhibit transcription 2.Bound transcription factors may also have an effect on the chromatin structure.

What are microarrays?

-They were the first technology that made analysis of transcriptome possible (1995). -A microarray is a hybridization-based technique that allows simultaneous analysis of thousands of samples on a solid substrate.

How is alternate mRNA splicing regulated?

-Via RNA-binding proteins -500-1,200 different RNA-binding proteins in humans -Proteins normally expressed in tissue-specific, developmental or disease-specific mannor.

What is a disadvantage of RNA-seq vs Microarray?

-Widespread expression distribution/range is a potential problem whereas it isn't for Microarray -This is the difference in the copy no between highest and lowest expressed transcript in the cell but depends on getting decent coverage (no of reads) to accurately sample a transcriptome making RNA seq very expensive

what are three important features of the genetic code

-almost universal- bacteria can translate human genes (useful in genetic engineering) -highly degenerate- most amino acids are coded for more than one codon -exceptions- one met (AUG) and trp codon -3 stop codons UAA, UAG, UGA -non random- differ only in third nucleotide, to reduce the effect of mutations

Zinc Fingers contain

-alpha-helix -beta-sheet -conserved cysteine residues -coordination to Zn

ENHANCERS

-cis-acting DNA sequences similar to promoter required to enhance the transcription -unique to eukaryotes -found in introns. 5-3' UTR -can affect gene expression in any direction -TRANSCRIPTIONAL ACTIVATORS BIND TO ENHANCERS TO ACTIVATE TRANSCRIPTION

proximal-promoter sequences

-cis-acting sequence precedes core promoter -specific to genes -binding site for transcriptional factors: activators and repressors -consensus sequences: GC box and CAT box

Homeotic(Hom) Genes

-first mutant observed in 1910 -drosophila -encode TF that hep reg. other genes for development -confers positional info along anterior-posterior axis in fruit flies -genes have common DNA sequence called homeobox that encodes heli-tuxen-helix domain -mutations can be lethal

helix-turn-helix

-first studied in drosophila -occurs in eukaryotes (Hox and Hom genes) -2 alpha helices separated by a beta turn -recognition helix contains aa contacting DNA in major-groove, sequence-specific -other helix contacts P groups in DNA backbone to strengthen binding, not specific

Zinc Finger Domain

-found in many eukaryote proteins -2-30+ per protein -interacts w/DNA via alpha-helices in major groove -30ish a.a. form structure w/tetrahedrally-coordinated Zn+2 ions

DNA-Binding Domains in TF's

-generally small regions of protein 60-90 a.a. -only a few of those a.a. actually make contact with the DNA -generally, reg. proteins must dimerize before binding

What is mRNA degradation?

-mRNA turnover in the cytoplasm is affected by several factors: 1.Length of poly(A) tail; longer gives more stability 2.Presence of 5'cap 3.Activity of exonucleases

Translocation

-movement of deacylated tRNA from the P site to the E site and movement of acylated tRNA to the P site, coupled to movement of the mRNA. -Mediated by EF-G, another GTPase

How does having multiple structures in UTR affect translation?

-multiple structures, SD and AUG not part of them- on, -small molecule binds -rearrange RNA structure -now part of structure- off= negative feedback

what occurs when tRNA is in the A/T position

-new tRNA enters sticks head out of R -checks if in correct position -easy to remove if wrong -if correct GTP hydrolysis occurs and moves to A/A site (and P/P site)

what is the endosome and what does it do?

-protein complex with exo and endonucleases -degrades RNA

What is fragile X syndrome?

-repeat CGG in 5' UTR -give long mRNA so can't make FMRP needed for synapse function- mental retardation -thin region X chromosome- prone to breaking

core-promoter sequences

-shortest cis-acting sequence required for initiation of transcription -close to transcriptional start site of a gene -TATA box usually ~40bp long -binding site for basal TFs and RNA polymerase

how are siRNA's used in research and medicine

-specific artificial siRNA's degrade mRNA via RNAi -gene knockdown- not very effective -siRNA vector can be inserted by lipid based transfection or viral transduction -look at weslem plot to compare against control

how do siRNA's regulate gene expression

-trigger dsRNA- cleaved by dicer enzyme- RNase type III -gives 21 to 23 nucleotide long ds siRNA -enters RNA induced silencing complex=RISC -seprates strands giving ss guide RNA -siRNA binds comp mRNA in cytoplasm -RISC cleaves via argonuate (slicer) RNase type III

Hox Genes

-vertebrate version of HOM genes -hox gene clusters on 4 separate chromosomes -each codes for TF's that reg transcription of genes for development in a specific temporal and spatial way -genes have common DNA sequence called homeobox that encodes heli-tuxen-helix domain -mutations can be lethal

Tell me some of the advantages of RNA-seq vs Microarray?

-£500 vs £1,200 -Unlimited dynamic range vs low-medium -No reference genome is needed vs needed -20-30gb data storage vs <2gb -Variable genomes aren't a problem vs a potential problem

draw out the recruitment of factors pathway

....

time course of the ordered recruitment of TFs

.....

tRNA synthetase, aminoacyl-adenylates

...........subjects..........to proofreading step

describe the steps of the process of splicing

1) U1 snRP binds to 5' splice site A branch point binds U2AF- helper protein which helps BBP to recognise branch point BBP binds to A base on branch point 2) BBP guides U2snRNP to bind to branch point giving A complex, BBP and U2AF dissociate 3) U4/U6.U5 triple snRNP bind at same time, have base pair interactions with each other. B complex rearrangements break giving energy (U4/U6) 4) lariat formation and 5' splice cleavage- U1 and U4 leave giving C complex. Branch point attacks 5' splice 5) 3' splice site cleavage join 2 exons- exon junction complex comes excise intron into lariat

Chromatin Immunoprecipitation (ChIP)

1) cross-link (two cell type conditions) then isolate chromatin 2) "break" chromatin into pieces 3) use specific antibody (these bind to proteins very specifically) to select chromatin pieces containing protein of interest 4) release DNA 5) use PCR to determine if a specific gene/ DNA sequence was bound to the protein; if nothing shows up on the bands, it means that the gene did not precipitate with the antibody; always use an input control; tells you what proteins are associated with DNA but can't tell you if its directly or indirectly bound

EMSA

1) incubate radioactively labeled ds DNA probe with protein 2) electrophorese under non-denaturing conditions 3) detect; if protein binds to DNA, it will run more slowly through a non-denaturing gel

What methods are there to detect single gene transcriptional changes?

1. Hybridization based -Northern blotting = transcription specific radioactive probes are used to identify specific mRNA species with an immobilized RNA sample 2.PCR based -Based on the ability of PCR to exponentially amplify initial differences in transcription no -Amplified products are viewed either in real-time (qPCR,A) or after the reaction (RT-PCR,B). 3.Reporter gene bases -Fusions of a promoter gene of interest with a reporter gene: b:glucuronidase (GUS), green fluorescent protein, luciferase. -Reporter activity is measured histochemically or by fluorescence or luminescence -Allows for detailed spatial and kinetic analysis of transcript accumulation.

what are the conserved intron sequences that are recognized for splicing at the 5' and 3' end? what is the consensus sequence at the poly A site?

1. Start of all introns (5' end): GU sequence that is bound by U1 snRNA 2. End of all introns (3' end): AG sequence; there is A branch point bound by U2 3. the Poly A site consensus sequence: AAUAAA sequence *although these are not sufficient to recognize introns for splicing, they are conserved sequences and play a special function in splicing.

What factors determine the presence of proteins in a cell?

1. Transcription 2. RNA processing 3. mRNA translation 4. mRNA degredation All leading to protein degradation

Describe how alternative splicing permits multiple proteins to be produced by splicing defects.

1. can retain or remove certain introns AND exons 2. can cause truncation or extension of the 3' or 5' ends, and can employ mutually exclusive exons. 3. If 5' splice site of an INTRON ever gets mutated, the intron itself may go unrecognized and remain a part of the final mRNA sequence, messing up the final protein.

What are the 3 steps involved in Transcription Termination

1. cleavage at the poly(A) site 2. addition of the poly(A) tail at the new 3' end 3. transcription termination downstream from the cleavage site

3 main repression mechanisms

1. competitive DNA binding 2. Masking the activation surface so coactivators can't bind 3. direct interaction with GTFs to block activator binding

List the functions of the 5' cap of the mRNA.

1. protect the 5' end of mRNA from nucleases. 2. makes RNA more recognizable to Cap Binding Complex, which facilitates further processing of the mRNA (in the nucleus). 3. Binds with eIF4E in the cytoplasm that helps initiate translation (facilitate transport to ribosomes). 4. when the cap is removes, signals mRNA to be degraded.

TATA box is located ___ of transcription initiation site

25-35 base pairs upstream

leaf clover

2D shape of tRNA

what is the genetic code

3 mRNA nucleotides is equal to one codon which codes for 1 amino acid or stop codon

coiled L

3D shape of tRNA

describe eukaryotic ribosomes

4 rRNAs made in nucleus assembled in cytoplasm

mRNA has

5'UTR, Kozak's, or the RBS sequence for translation of the protein, start and stop codons, 3'UTR with ARE sequence (a signal for polyA tail synthesis and a polyA tail (post-transcriptional gene regulation)

what are the ribosomal subunits in bacteria

50s - 5s rRNA (conserved) 23SrRNA+ 34 proteins 30s- 16sRNA +21 proteins

more than __ must be sequentially recruited to transcribe a human protein coding gene

60

what are the ribosomal subunits in eukaryotes

60s large subunit-5srRNA, 28sRNA, 5.8s rRNA, 49 proteins 40s small subunit- 18s RNA+ 33 proteins

final result of initiation

80s ribosome with mRNA and met~tRNA at the P-site (peptide site of the ribosome)

ribosomal elongation sites

A- first spot (on deck to be added) P- second spot (amino acid to be added) E- site (exit site)

what are antisense oligonucleotides

ASO degrade RNA and block translation edit spicing similar mechanism to siRNA but good bioavailability

RNA Polymerase

An enzyme that starts (catalyze) the formation of RNA by using a strand of a DNA molecule as a template

Exon junction complex

An exon junction complex (EJC) is a protein complex which forms on a pre-messenger RNA strand at the junction of two exons which have been joined together during RNA splicing. The EJC has major influences on translation, surveillance and localization of the spliced mRNA.[1] It is first deposited onto mRNA during splicing and is then transported into the cytoplasm. There it plays a major role in post-transcriptional regulation of mRNA.

DSIF

Bind to RNA Pol II. Pause transcription of a gene after the transcript has been initiated. (2)

coactivators with histone acetyl transferase activity

CBP and p300

EF-Tu

Charged tRNAs are not free to diffuse into place but are escorted by _____ to the ribosome

DNA can be damaged by

Chemicals and ultraviolet radiation ( Uv from the son )

Describe step 1 of elongation.

Codon recognition: incoming tRNA anticodon carrying amino acids pairs with mRNA in A site

TF2D

Composed of TBP (TATA binding protein--bends the DNA) and TAFs (2D associated factors--recognize non-TATA core promotors)

B DNA

DNA coated in DNA binding transcription factors ~ 10% of protein-coding genes have affinity for these TFs Sequence specific info about DNA is accessible to these proteins even in double-stranded form Contact and non-covalently interact with major and minor groove (more contacts with former b/c more space and more residues available for contact)

regulation by activators/repressors bound at distant sites involves

DNA looping architectural DNA-binding proteins facilitate bending of DNA

ADP-ribosylation

Diphtheria toxin catalyzes the ___ of eEF-2, which blocks protein synthesis

Acronym for RNA polymerase II pre-initiation complex

DnA Bending 2 Facilitate Efficient Helicase

3 binding sites from mRNA in ribosomes

E, P, A site

Stop codon

Elongation ends when a ____ is reached

eIF-2

Eukaryotic initiation factor 2 when activated by GTP it binds to charged tRNA that carries the initiating methionine **only met~tRNA is recognized by eIF-2 (met corresponds to AUG start codon)

What are the various alternatives for adding the poly A tail?m

Example: IgM (immunoglobulin) has two different forms 1. If poly A site is chosen more upstream: -->secreted form mRNA plasma cells and has different function 2. If poly A site is chosen more downstream: -->secreted membrane form mRNA B cells and has different functions **alternative addition of poly A tails changes the sequence of the 3'UTR.

Accomodation

Excess binding energy from recognition of cognate base pairs at the minor groove is used to induce conformational changes essential for GTPase activation; small subunit interacts with minor groove of the codon-anticodon helix only if it's a cognate match

1. intramolecular interactions 2. protein-protein interactions 3. post tranlational modification

Final structure of proteins is dictated by __1__ due to primary structure and by other factors, including __2__ and __3__

5' untranslated region or 5'UTR

From the beginning of the mRNA to the start codon

step 3 termination

GTPase is activated and hydrolysis of GTP to GDP leads to a release of the factors and release of the new peptide chain

In eukaryotic gene regulation

Gene expression is regulated during transcription and RNA processing, which takes place in the nucleus. Protein translation takes place in the cytoplasm. Further regulation may occur through post-translational modifications of proteins.

Codon

In DNA and messenger RNA, a three- nucleotide sequence that encodes an amino acid or signifies a start signal or a stop signal

E site

In translocation, Uncharged tRNA is displaced to ___, from which it is released

Pyrrolysine

It uses the UAG stop codon and a hairpin loop similar to the SECIS element.

describe tRAN tertiary structure

L shape amino acid maximum distance from anticodon hinge region made up of D loop and TpsiC loop

Transcriptional repressors

Like activators, include a DNA binding domain and another domain for repression

Accuracy of adenylate formation is...

Low

Antibiotics

Many ____ block translation

degenerate

Most amino acids have more than one codon

An error changes the DNA

Mutation

1) TFIID, TATA 2) TFIIA, TFIIB 3) TFIIF 4) TFIIH, TFIIJ, TFIIH

Order of TFIIX Binding 1. _________ binds to _________ 2. _________ and _________ bind next 3. _________ binds accompanied to RNA Pol 2 (Cannot begin transcription yet) 4. TFIIF, _________, and _________ bind; _________ phosphorylates the tail of RNA Polymerase

what tRNA's are bound in the post translocational site

P and E site

EF-Tu GTPase (corresponding GEF is EF-Ts)

Peptide bond Formation: -Entry of aminoacylated tRNA to A site, mediated by ___

Describe step 2 of elongation.

Peptide bond formation: polypeptide detaches from tRNA in P site and attaches to A site using a peptide bond

23S rRNA

Peptidyl transferase activity appears to be that of the _____

Compare and contrast a pre-mRNA with a mature mRNA.

Pre-mRNA is significantly longer (because it has all its introns), does not have a 5' cap (has a triphosphate group instead), and does not have a polyA tail.

Aminoacyl-tRNA synthetase: pre-transfer vs post-transfer editing

Pre-transfer editing - Dissociates the enzyme bound Aminoacyl-adenylate Post-transfer editing - De-acylates noncognate amino acids from the tRNA E = Aminoacyl-tRNA synthetase enzyme AA = Amino acid AA-AMP = Aminoacyl-adenylate E∙AA-AMP = Enzyme bound Aminoacyl-adenylate AA-tRNA = Aminoacyl-tRNA

Mass Spectrometry

Preferred method for detecting and resolving specific proteins in cell extract

synthesis

Protein folding begins during _____

What are the release factors in E.coli

RF1- UAA and UAG RF2- UAA and UGA

tRNA

RFs have domains that may mimic ___, suggesting how they work

H20 (hydrolysis rather than transfer to amino acid)

RFs induce transfer of the peptide to _____ by peptidyl transferase reaction, releasing the nascent peptide (pro)

How do miRNA's regulate gene expression

RNA II transcript undergoes cropping by micro processor complex- RNase III- drosha, form pri-microRNAs travel from nucleus to cytoplasm dicing by dicer protein giving pre-microRNAs -one strand degrades RISC complex with ago and proteins bind to microRNA which is 21-23 nucleotides long -extensive match or less extensive match outcome

What is RNAi

RNA interference short interfering RNA= siRNA nad microRNA's= miRNA regulate gene expression by binding to complimentary RNAs

basal transcription factors (TFs)

RNA polymerase can not bind to promoter correctly -TFs bind to TATA box to form pre-initiation complex

Ribosomal RNA

RNA that is in the ribosome and guides the translation of mRNA into a protein ; also used asa molecular clock

tRNA aminoacylation reaction

Reaction energy for Peptidyl Transferase Reaction is derived from the cleavage of the high energy ester linkage formed by the ____ reaction

RNA

Ribonucleic acid, a natural polymer that is present in all living cells and that plays a role in protein synthesis

Fundamental (original) wobble pairing hypothesis

Shows which bases can pair together in the wobble position

Gel Mobility Shift Assay (EMSA)

Start w/ polyacrylamide gel Radioactively label DNA fragment (can label w/p32) Mix DNA with cell extract containing various proteins, run gel DNA ending up closest to gel wells is bound to protein (thus slower mobility) **Use native gel b/c don't want to denature either the DNA fragments or proteins (even though charge of proteins plays a role, this impact is overshadowed by mobility changes after protein-binding) **Can run gel after fractionation of cell extract via differing salt concentrations

Proteome Readouts (not used much anymore, explain how to create)

Start w/lysate of cells with just proteins Subject lysate to 2D PAGE, running it through native gel with different pH at each end to separate proteins by charge Run through SDS PAGE to separate same proteins now by molecular weight

Describe elongation termination.

Stop codon stops translation at A site

__ binds to the TATA box

TBP

RNA pol2 forms a complex with:

TBP, TFIID, TAFs

Phylogenetic Footprinting

Take sequences from closely-related species to analyze regions of high conservation--these regions are candidates for TF binding Use other lab techniques to figure out what protein would bind to these candidate sequences

polycistronic

The mRNA from an operon

Define pre-mRNA

The nuclear transcript that is processed by modification and splicing to give an mRNA.

anticodon

These bases pair with the 3 bases of the codon on mRNA during translation.

A site P site E site

Three sites of elongation and the order the tRNA travels through them

tRNA binding, peptidyl transferase activity, and translocation

Translation Elongation is repeated cycles of ___, ___ and ____

stop codons

UAA, UAG, and UGA, ending code for the CDS

Termination (stop codons)

UAG, UAA, UGA

Dna replication is

Very accurate

eEF-1β

___ is euk analoge to EF-Ts

EF-1α (called EF-Tu in bacteria)

a G protein elongation factor that hydrolyzes GTP

proteasome

a large, multi-subunit molecular machine that degrades proteins All of its subunits are proteins, has a cylindrical shape with 3 parts

Unlike prokaryotes, eukaryotic cells possess

a nucleus and a nuclear membrane

isopeptide linkage

a peptide bond, but it is not part of the main polypeptide chain

inosine

a purine that can base pair with C, A, or U. made by deaminating adenosine

3' terminal ribose

aa attaches to.........of tRNA by covalent bond in aminoacylation

TFIIF

accompanies RNA Pol II as it binds to promoter

puromycin

antibiotic that mimics aminoacylated tRNA as a polypeptide chain terminator

exon

any sequence in DNA that ends up in the messenger RNA

introns

any sequence in DNA that is transcribed but spliced out of the primary transcript.

when does initiation start

at AUG recognised by initiator tRNA carrying met the initiator tRNA is different than normal and met is removed later

How does translation repressor protein and UTRs interact?

bacteria- 5' UTR has structure and shine dalgarno sequence before AUG=on -translation repressor protein binds to s.d so ribosome can't bind

degenerate

because genetic code is.....each aa can be carried by more than one tRNA

EF-1

brings charged tRNA to the A site of the ribosome

what is a point mutation

change in one nucleotide which may or may not have large effects

what is the spliceosome

complex of large machinery made of 5 small nuclear RNA's and proteins

Shine Dlegarno sequence

consensus sequence in prokaryotes that acts as the ribosomal recognition binding site in eukaryotes this function is carried out by EIF4

elongation

consists of decoding, accommodation, peptide bond formation, and translocation

example is TATA box, TFIIB recognition element, initiator, downstream promoter element and they specific the site of transcription initiation

core promoter motifs

promotors contain:

core promoter motifs and recognition site for sequence specific DNA binding activators

7nt loop containing dihydrouridine

d arm ends in

TFs bind to DNA as

dimers bc ds DNA

Difference between promoter proximal elements and enhancers

distance to promoter only

Repressors in eukaryotes

do not sterically block binding to the promoter

Diphtheria Toxin

eEF-2 is a ___ target

3' untranslated region or 3'UTR

end of the stop codon to the end of the mRNA

aminoacyl-AMP + tRNA --> aa-tRNA + AMP

equation for step 2 of aminoacylation

non-ambiguous code

everything codes for something, multiple codons and tRNA per amino acid

AspRS in yeast

example of aminoacyl-tRNA synthetase

Genes with only sites for GTFs and RP II

exhibit basal transcription levels

proteins associated with ribosome

fold on surface and contain long unstructured parts that often have positive aa to stabilize RNA negative phosphate backbone

what RNA gets its introns removed?

heterogenous nuclear RNA in nucleus

symmetrically binds 2 tRNA, dictated by induced fit not sequence

how AspRS binds tRNA

ribosome

in Peptidyl Transferase Reaction note change of conformation such that tRNAs shifted across ____ (anticodons still in A and P sites_

Chromosome in eukaryotes are located

in the nucleus therefore the transcription occurs in the nucleus

cycloheximide

inhibits: eukaryotic translation mechanism: inhibits peptide transferase (60S)

tetracyclines

inhibits: prokaryotes mechanism: interacts with 30s ribosomal subunit/inhibit binding of incoming AA-tRNA to A site

Puromycin

inhibits: prokaryotic and eukaryotic translation mechanism: premature termination

what is the issue with using siRNA, miRNA and synthetic mRNA

low bioavailability don't cross cell membrane easily

Circularized mRNA

mRNA circularizes prior to the initiation of translation, connecting the 3' and 5' Neds possible to ensure that only completely process mRNA's are transcribed (5' cap and poly A tail must be present for circularization)

GTPase

mechanochemical molecular switch that propagates change in conformation of protein and undergoes change after hydrolysis stimulated by GAPs to dissociate and allow tRNA to move into ribosome and establish proximity at A and P site; this contributes to the rate and fidelity of protein synthesis

Proteins that regulate trx are

modular

eRF-1

recognizes all termination codons (euk)

eRF1 and eRF3 (step 2)

release factor 1, release factor 3 and GTP bind to the stop codon

Regulatory DNA sequence elements

required for effective initiation, both near the promoter and very far

what does chemical modification of the RNAs

snoRNAs process and modify other RNAs mostly rRNA

temporal regulation

specific proteins are required at specific times only. cells do not need all the portend at all times

guanosine nucleotide exchange factor (GES) protein

switches GDP with GTP for G proteins

RNA polymerase III

tRNA

Describe the basic process of elongation.

tRNA pairs with each codon, adding an amino acid to growing polypeptide

initiator methionine

tRNA unlike the elongator methionine that initiates transcription by binding in the P site

70S ribosome

the assembled ribosome in prokaryotes. It is smaller than the eukaryotic ribosome

Pre-initiation complex

the cap is bound by an IF factor and the tail by PABP that interact with each other via a complex of other factors causing the DNA to circularize and helps put PIC onto circular template; closed loop complex that leads to circularized polysomes

80S ribosome

the combination of the 60S and 40S subunits

60S subunit

the large subunit of a ribosome. It has 3 RNA molecules and 46-50 polypeptides

ribozyme

the ribosomal RNA catalyzes the chemical reactions of protein synthesis.

SP1

transcription factor that binds to GC Box in promotor

EF-G, aka translocase

translocation is mediated by ___, aka ____

covalent

type of bond that links aa to tRNA

Newly synthesized mRNA is

unprocessed, consists of alernating introns and exons and referred to as heterogeneous nuclear RNA, or hnRNA

General and specific DNA affinity chromatography

used a DNA matrix column with increasing salt washes to elute proteins of different binding levels

G proteins "OFF"

when a G protein hydrolyzes GTP into GDP

Where is RNA modified

• RNA is modified in the nucleus by additions to the 5' and 3' ends and by splicing to remove the introns.

Release Factors (RFs)

____ enter the A site when a termination codon is reached

translocation

____ is a shift of the ribosome along the mRNA by one codon

Peptidyl transferase

____, an activity of the 23S rRNA of the large subunit, catalyzes peptide bond formation that transfers growing peptide to A site

What happens when there is an "ABSENCE OF LIGAND"?

*Imagine a protein dimer composed of two identical subunits, each with a LIGAND BINDING SITE. -In the absence of ligand, both sites are in a "closed" state that has LOW AFFINITY for LIGAND.

Nomenclature

*Prefix* - Indicates amino acid the tRNA species is aminoacylated with - No prefix means the tRNA is uncharged *Superscript* - Indicates which family of tRNA isoaccepters this particular tRNA belongs to - tRNA can belong to 1 of 20 families There is one family per amino acid *Subscript* - Indicates which specific isoacceptor species this tRNA molecule is - Subscript is sometimes numerical, sometimes the anticodon Example Leu-tRNALeu3 means the aminoacylated form of the 3rd leucyl-tRNA isoacceptor

Identity element locations

*Primary identity elements* - Anticodon (position 34, 35, 36, mostly the last two) - The identity element that the aminoacyl-tRNA synthetase will recognize will be in the anti-codon - True for 16 out of the 20 tRNA families *Secondary identity elements* - Acceptor stem - Exp: G-U base pairing between position 3(G) and 70(U) in the acceptor stem is necessary and sufficient for the alanyl-tRNA synthease to recognize that tRNA as being in the alanine family and to aminoacylate that tRNA with alanine - Anticodon is irrelevant for the alanyl-tRNA synthetase

tRNA "identity" elements and families of isoacceptors

- Allow aa-tRNA synthetases to identify which tRNAs are in their family - Identity is critical because once the aa is attached to tRNA, it is committed to protein synthesis in response to cognate (family) codon to the tRNA - Shown experimentally with Raney nickel (described in lecture 5)

Aminoacyl-tRNA synthetases edit mistakes

- Aminoacyl-tRNA synthetases have proofreading mechanisms that prevent non-cognate (non-family) amino acids from getting onto the tRNAs - Editing can occur pre-transfer or post-transfer of aminoacyl-adenylate to the tRNA

Acceptor stem

- Area at the 3' end of the tRNA where 3' and 5' end bind *5'-CCA-3' overhang* - Universal component of all tRNAs - Overhangs from 3' base pairing region of acceptor stem - Serves as the sight of attachment for amino acids -- amino acids to be incorporated into the growing peptide chain attach to the 3' end of the terminal Adenosine

Schematic representation of the three-dimensional structure of tRNA

- Circles indicate the positions of nucleotides, the size of which is proportional to the documented frequency with which they are involved in recognition by aminoacyl-tRNA synthetases - Anticodon includes nucleotides 36, 35, and 34 (wobble position) - The acceptor stem refers to the helical structure formed by base pairing of bases 1 to 7 with bases 72 to 66 respectively - Students are responsible for principles, not specifics

Generalized structure of tRNA

- Cloverleaf in 2-D - "L-shaped" in 3-D

Unique features of initiator tRNA

- In order to initiate translation, a tRNA must first enter into the P site (peptidyl) - Three G-C base pairs in the anticodon stem - Allows tRNA to bind at the P site binding begin initiation - Any tRNA that has the three G-C base pairs in the anticodon stem is going to go into the P site

Wobble

- Indicates the flexibility in the tRNA decoding of codons - Greater flexibility in base-pairing between the 3rd codon and the 1st position of the anticodon

Variable loop

- One of the factors that distinguishes one tRNA from another tRNA - Can have expansion of nucleotides in the variable loop

tRNA processing

- RNase P conducts tRNA processing yielding active (mature) tRNA form - Contains an RNA component responsible for catalysis, as well as protein components

Inosinic acid

- Rare modified base (rare in bacteria) - Has great flexibility in the anti-codon pairing with codons in the wobble position

Incorporation of selenocysteine in protein

- Selenocysteine is an unusual case of recoding where the signals in the mRNA that would normally lead a stop in protein synthesis, due to a UGA stop codon, instead is reprogrammed to allow the incorporation of selenocysteine - Selenocysteine is incorporated in response to a UGA stop codon - Biosynthesis and incorporation of selenocysteine into proteins of Escherichia coli compared with that of the 20 standard amino acids - Stem-loop (hairpin) structure - The discrimination of the UGA (selenocysteine) from UGA (stop) codons is mediated by a stem-loop structure 3' to the UGA codons in the mRNAs - Students are responsible for concepts only

Extensive use of modified bases in tRNA

- Some of the modified bases function in decoding - Modifications affect codon-anticodon interaction - Provide specificity for which codons can be decoded by the tRNA bearing those respective modifications - Modifications increase the area of the molecule that is hydrophilic - Allows for increased interaction in solution with other molecules - Modifications are all post-transcriptional

Aminoacylation

- The addition of the amino acid to the terminal adenosine at the end of the CCA sequence located at the 3' end of the tRNA molecule - Note that the aminoacyl-tRNA link is a high energy bond, which is the source of energy for peptide bond formation on the ribosome

mutations

- missense changes amino acid - silent causes no change - frameshift occurs when the third position is removed or changed and creates a new reading frame usually resulting in an early stop

recycling

- prokaryotic release factors bound to GTP that break apart ribosome by GTP hydrolysis; initiation factors bind and prevent reassociation - eukaryotic dissociate via ATP hydrolysis and initiation factor binding

What are the mature rRNA?

-- 18S -- 5.8S -- 25S

What is the poly A signal?

-- AAUAA

what tRNA modification occurs in all species?

-- CCA replaces the U's at the 3' end'

What does RNA processing include?

-- Covalent modification of bases within an RNA transcript -- Removal of nucleotides from primary transcript -- Addition of bases not encoded in the gene to the ends of pre-RNA transcripts

What does endoribonucleases do?

-- Digest the the first primary transcript

What are ribosomal RNA?

-- Do not encode proteins -- Structural RNAs forming the ribosome. -- Catalyze the peptidyltransfer reaction during translation.

When are RNA molecules modified?

-- During or after transcription

What is ESE?

-- ESE: exonic splicing enhancer

What is ESS?

-- ESS: exonic splicing silencer

What does Cleavage Stimulatory Factor (CStF) recognize?

-- G/U rich sequence

What are tRNAs?

-- are involved in protein translation -- Activated tRNAs carry amino acids covalently linked to their 3'-end

How do mutations often cause diseases?

-- by disrupting splicing

What is polyadenylation considered?

-- co-transcriptional

How are The RNA polymerase II transcripts are extensively processed?

-- co-transcriptionally

What do self splicing introns do?

-- interrupt the ribosomal rRNA genes in chloroplasts (Group II introns) and some microbial eukaryotes (Group I introns in Tetrahymena)

What is primary RNA transcript?

-- is generally referred to as "RNA processing"; typically this begins "post-initiation of transcription", but prior to termination.

What is polyadenylation?

-- mRNA 3'-End processing

What are the ribosomes that form the ribosomes?

-- rRNA

How do you recognize an intron?

-- starts with 5'-GU -- eds with 3'-AG

What does self splicing mean?

-- the introns excise themselves and ligate the rRNA without the assistance of protein enzymes.

What synthesize the poly-A tail?

-- the poly-A polymerase enzyme (PAP)

What does mRNA splicing involve?

-- two consecutive *transesterification* reactions

How is biological interpretation done?

-Data processing and various stats and analytics are done producing a list of genes and can contain several hundred genes. -But list of 'interesting genes' must be interpreted (the hard part) -The gene Ontology Consortium is a major collaborative initiative between bionformatics and people with specialised scientific knowledge have developed 3 structured vocabularies (ontologies) that describe gene components and molecular functions in a species independant mannor with the goal of simplifying gene description. e.g. P450 gene CYP6P4 = oxidation reduction process &may have many molecular functions such as iron ion binding and electron carrier activity etc.

What is an example of a RNA seq?

-Deep sequencing of Pyrethroid resistant bed bugs reveals multiple mechanisms of resistance with a single population. -Adelman et al. (2011) 2.5 million reads. Cytochrome P450 carboxylesterase genes over expressed in resistant strain. Metabolic resistance to insecticides. Resistant insects also had mutations in their sodium channels target site resistance.

What is an example of hormonal control of gene expression?

-Glucocorticoid (glucose + cortex + steroid) synthesized in the adrenal cortex. -Glucocotocoid stimulates down turn in immune activity -Produces expression of anti-inflammatory proteins -Repress the expression of pro-inflammatory proteins.

What is significant structural features of chromatin for transcription?

-Histones are proteins involved in tightly packing DNA into nucleosomes. -But tightly packed DNA isn't available for transcription of genes. -Chromatin remodelling allows transcription proteins to access DNA -Enzymatic modifications of histone proteins produce chromatic remodelling: Acetylation; Methylation; Phosphorylation; Ubiquitylation. -Structural changes to chromatin can be long lived. -Methylation of DNA cytosine bases also effects both chromatin structure which is another factor that determines long-term expression patterns which is relevant for epigenetics.

What is the situation for cytosines and histones when the gene is 'switched off'?

-Methylated cytosines -Deaccetylated histones

What is RNA-seq?

-Next generation sequencing of the transcriptome acting as a modern alternative to microarrays. -The technology includes Roche 454 sequencing; Illumnia MiSeq and Life technology Ion Proton. (see video on how it is done)

What is Affymetrix HGUI33 + 2 chip?

-Probsets can be designed instead of using a single probe per gene. -It interrogates the human transcriptome ~2000 genes in the genome, ~54,000 probesets, some probesets interrogate different regions of the same gene whereas some interrogate non-genic regions.

What are Short interfering RNAs (siRNAs)?

-Processed from double-stranded RNA -Knock-out mice mice lacking the gene for Dicer die early in embryonic development.

What is the basic structure of a Eukaryotic cell?

-Promoters are located upstream and on the same DNA strand as the transcription start site. -A gene may have 1 or more promoter. -Promoters are the regions where transcription factor proteins bind (necessary to initiate transcription) -The promoters of many eukaryotic genes (approx 25% of human genes have a 'TATA' box) 1.DNA sequence 5'TATAAA3' or very similar 2.Usually located 25-35 base pairs upstream of transcription start site 3.Site where 'TATA binding protein (TBP)' binds during initiation or transcription

What are enhancers?

-Short (50-1500bp) regions of DNA -Distal from gene sequence (upto 1Mbp away) but spatially close. -Located upstream or downstream and either in forward or reverse direction from gene. -Enhances bind activator proteins that affect transcription factor or RNA polymerase II binding. -Enhancers enhance activity of promoters but can't substitute for them -Silencers are like enhancers but repress transcription

What are MicroRNAs (mRNAs)?

-Small non-coding RNA molecule of ~22 nucleotides ->1000 mRNAs: identified in humans

Which requires the "HIGHER LIGAND" concentration?

-The FIRST binding thus requires a HIGHER ligand concentration than the SECOND LIGAND.

How are microarray results processed?

-The microarray is optically scanned -Ratios of red to green are computed for each spot (for 'feature') and converted to log scale (easier to link log ratio to fold chain in expression) -Cy3 =control (green) Cy5=experimental sample -Cy5>Cy3 higher expression in sample 2 -Cy5<Cy3 higher expression in sample 1 -Cy5=Cy3 equal expression in both samples -After calculating amount of repression or induction for each gene, the data is further 'sorted' computationally using algorithms e.g. Hierarchical clustering

What is transcriptomics?

-The transcriptome is the set of all mRNAs present in certain cell, tissue organs etc. at a specific time or under a specific set of conditions. -A transcriptome is extremely dynamic and can be much more complex than the genome that encodes it. -We can study different tissues at different times for an insight into where and when each gene is active within cells and tissues of organisms. -We can continue the number of transcripts to determine the amount of gene expression (activity) in a certain cell or tissue type -Via comparison of transcriptomes we can further understand what is a specific cell type, how it normally functions and how normal level of gene activity may reflect or contribute to phenotype (e.g. drug resistance, disease etc.) -Most genes are not currently associated with a function via searching through the transcriptome database researches can gain an insight to all the tissues in which a gene is expressed, providing clues to its possible functions.

What is Hierarchical clustering?

-This is when genes with similar expression patterns are grouped together and are connected by a series of branches (clustering or dendrogram) -Top=relations between samples e.g. patients or time points. -Side=Relations between genes

What is Basal transcription?

-Transcription factors are sequentially recruited close to the gene (e.g. TBP, TFIIA). -RNA polymerase II is a multi-subunit enzyme that forms a complex with the transcription factors. -Phosphorylation of RNA polymerase II (by the kinase TFIIH) is necessary for initiation of transcription.

What is the situation for cytosines and histones when the gene is 'switched on'?

-Unmethylated cytosines -Accetylated histones

Assembly of RNA polymerase II pre-initiation complex

1) TF2D binds 2) TF2A binds in vivo only 3) TF2B binds 4) RNA polymerase II enters pre-formed with TF2F and binds 5) TF2E binds 6) TF2H binds (brings in helicase and phosphorylates CTD so that Poly II can zip along) 7) transcription starts and all general factors are released except for TBP because it can help start the process over

What are the three biggest base modifications to tRNAs?

1) replacement of U residues at the 3′ end of pre-tRNA with a CCA sequence, which is found at the 3′ end of all tRNAs; 2) addition of methyl and isopentenyl groups to the heterocyclic ring of purine bases, and methylation of the 2′-OH group in the ribose of any residue (impacts endo conformation of the ribose); 3) and conversion of specific uridines to dihydrouridine (D), pseudouridine (Ψ), or ribothymidine residues.

DNAse I footprinting

1) synthesize specific DNA sequence via PCR then radioactively label (32P) ds DNA at one end 2) incubate DNA with protein sample 3) add limiting amount (so that you make only one cut per molecule) of DNAse I 4) isolate DNA 5) denature and separate by electrophoresis 6) detect labeled DNA fragments; you test one sample with protein and one without; DNAse I will cut everywhere that isn't protected by being bound to protein; from this experiment you learn whether or not the protein binds, where it binds, and the size of the footprint

What are the 4 ways the 5' and 3' UTRs control mRNA translation

1) translation repressor protein- turn off 2) RNA 'thermosensors' - turn on 3)multiple structures 4) antisense RNA

Describe the steps of translation initiation

1)small ribosomal subunit, initator tRNA bound to P site and eIF2 helper protein- form pre initiation complex 2) mRNA enters- helper proteins bind, eIF4E- 5'cap, EIF4G- polyA binding proteins 3) PIC binds, scans for first AUG- use ATP 4) initiation comp forms when base pairs 5) eIF2 and other initiation factors dissociate, large subunit binds, complete ribosome formed, met tRNA in p site

What are the 3 major steps in pre-mRNA processing steps? And why is it important

1. 5' caping: addition of modified G residue 2. splicing: removal of introns and ligation of exons to assemple the open reading frame 3. 3' polyadenylatoin: cleavage and addition of Adenine (poly A tail) at the 3' end This step of processing is important because it modified mRNA to enhance nuclear export and translation and to prevent degradation Note: these processing starts while RNA is being transcribed.

Four stages of protein synthesis

1. activation of amino acid 2. chain initiation 3. chain elongation 4. chain termination (2-4 are all ribosomal)

three sources of error in translation

1. aminoacyl tRNA synthetase uses wrong amino acid substrate 2. aminoacyl tRNA synthetase uses wrong tRNA as substrate 3. ribosome uses wrong tRNA

Decoding

1. rapid and reversible binding of EF-Tu and aminoacyl tRNA 2. stabilization of cognate aminoacyl tRNA that was selected based on ability to pair with mRNA; if there is enough energy due to bonding the small subunit will change conformation that propagates to GTP 3. GTP hydrolysis 4. Dissociation of EF-Tu that slows down to sample amino acid if it is a near cognate match, it may still fall out after GTP hydrolysis due to low energy that cannot overcome torstional strain

What are the two key elements that are recognized by base pairing during splicing

1. the 5' splice site is 1st recognized by base pairing to the U1 snRNA 2. there is an A branch point recognized by base pairing to the U2 snRNA --the 3' splice site has a U2 associated factors (U2AF), which is recognized by the U2 snRNA **mutation in the U2AF (disruption in the splicing) is associated with certain types of leukemia..

Describe the steps involved in the cap enzymatic process

1. triphosphatase: removal of the gamma phosphate note: addition of cap structure that replaces the 5' triphospate with modified G residue occurs in reverse ( 5'--5' triphospate linkage and NOT the 5'--3' phosphodiester linkage) 2. guanylytransferase: addition of the guaninetriphosphate and cleavage of the diphosphate 3. Guanine 7 methyl transferase: addition of methyl to the Guanine at 7' position --Second methylation occurs at the 2' end of the first base. (Helps ID b/n host RNA and viral RNA during viral attack)

What is a less extensive match with miRNA's

18-16 nucleotides the same- incomplete match mostly in humans bind mRNA at 3' UTR- regulatory elements -rapid repression of translation eventual degradation using the same mechanism as functional mRNA break down via poly A tail

how are ribosomal genes cleaved to give a the ribosome subunits

18S rRNA- small ribosomal subunit 3.8S rRNA, 28S rRNA- large ribosomal subunit (plus 5S rRNA which is made by RNAPIII in nucleoplasm)

What are the steps for mRNA 5'-end formation (capping)?

1: remove gamma (γ) phosphate 2: *Guanylyltransferase attaches a G residue via 5'-5' triphosphate linkage to 5' end* 3: methylation at position 7 of G 4: commonly also have methylation at 2' hydroxyl group of ribose for first two nucleotides

Translation: Initiation

1st step, uses an initator tRNA to start translation.

Transcription Factors (structure, specificity representations, binding formations)

2 domains (bipartite): DNA-binding/recognition domain + activation/suppression domain (sometimes more than one of each) "Logo" representation: illustrates the residues in TFs that make up the specificity for DNA recognition -- obtain via ChIP Seq with tagged TF bound to target regulatory sequence, then sequence the stretches of DNA bound to the TF Dimer binding formation increases specificity and stability (greater affinity) in binding to DNA

Ribosomes

2 subunits; RNA and protein comprise the structure - prokaryotic is a 70S protein with a 50S large and 30S small subunits - eukaryotic is 80 S with 60S and 40 S subunits interface between the large and small subunit is where tRNAs bind and catalysis occurs

Anticodon

A region of a transfer RNA molecule that consists of a sequence of three bases that is complementary to an messenger RNA codon

aminoacyl-tRNA synthetases

A set of 20 enzymes that are used to "charge" (that is, attach) the tRNA with the proper amino acid.

what are the three sites of a ribosome

A site- aminoacyl-tRNA- where newly charged tRNA's enter P site- peptidyl-tRNA- growing peptide attached to E site- exit

why is no energy consumed in transpeptidation

AA position in a way that attack occurs easily -rRNA obstructs so water cannot bind as causes premature termination amino acid in A site amino group attacks polypeptide carboxyl group bound to P site

activation of tRNA

ATP + amino acid via tRNA synthetase creates an activated adenylated amino acid and PPi that is broken down into 2 Pi + heat driving the reaction forward tRNA + adenylated amino acid via tRNA synthetase create AMP and aminoacyl tRN class I transesterify to move amino acid 2' to 3' position

What is an example of situ-synthesized Oligonucleotide Microarray?

Affymetrix Gene Chip with ~10^6 'features'

Puromycin

Analog of aminoacyl tRNA that accepts peptide from the P site; acts as a chain terminator in both pro and euk translation

Wobble concept

Antiparallel pairing with codon Wobble with 3rd codon position 3rd position of the codon pairs with the 1st position of the anticodon

Acceptor stem bacteria vs eukaryotic stem

Bacteria - In bacteria the CCA is encoded in the gene - The tRNA undergoes modifications to become mature Eukaryotic cells - In eukaryotes the CCA is not encoded in the gene - Has to be added post-transcriptionally - Means that primary tRNA transcript is nonfunctional - CCA adding enzyme - Adds CCA to the tRNA in eukaryotes - Bacteria also have the CCA enzyme, which adds CCA in the event that it is removed during processing - In the 3-D structure the anticodon and the terminal CCA are far apart

Negative Elongation Factor

Bind to RNA Pol II. Pause transcription of a gene after the transcript has been initiated. (1)

hydrolysis

Binding of EF-Tu to the acceptor arm of tRNA protects the AA-tRNA linkage from _____ before entry into A site, minimizes energy waste

Erythromycin

Binds irreversibly to a site on the 50S subunit and blocks the tunnel by which the peptide leaves the ribosome, thereby inhibiting termination/translocation.

Streptomycin

Binds to the 30S subunit and distorts its structure, interfering with the initiation of protein synthesis

Describe the Machinery required for both cleavage & polyadenylation

CPSF - binds to polyA signal AAUAAA PAP - adds the poly(A) tail; is recruited by CPSF Symplekin - part of a larger complex that includes CstF and CPSF; - It helps to assemble or stabilize the CstF complex and hold the entire cleavage/polyadenylation machinery together

CREB activator example

CREB (cAMP response element binding protein) requires the CBP (CREB binding protein) coactivator to activate transcription

Start codon

Codon that signals to ribosomes to begin translation; codes for the first amino acid in a protein

Stop codon

Codon that signals to ribosomes to stop translation

Helix-loop-helix

DNA Binding domain with two helices and a loop, forms a dimer, similar to leucine zippers

Nucleases proofhead

DNA and repairs errors

transcriptional activators have separate domains for

DNA binding and activation

Homeodomain

DNA binding domain with 3 helices and a helix-turn-helix motif, structurally conserved

Zinc finger

DNA binding domain with a helix, beta sheet, and ion that forms a dimer with rotational symmetry

Leucine zipper

DNA binding domain with an amphipathic alpha helix that forms a dimer

EF-Tu:tRNA complex

EF-G: The TranslocaseStructural Mimic of ____

ester linkage

EF-Tu protects the ____ of the amino acid to the tRNA

proofreading

EF-Tu provides ____ function of codon-anticodon interaction -another contribution to translational fidelity

step 2 elongation

EF1 bound to GTP brings next charged amino acid tRNA to the A site (acceptor)

step 4 elongation

EF2 bound to GTP is used to move contents of P site to E site and contents of A site to P site 2 GTP per amino acid added steps are repeated for each amino acid added in the growing peptide

folding

Following termination and release, proteins complete _____

Transfer RNA

Form of RNA that brings amino acids to ribosomes during protein synthesis

Messenger RNA

Form of RNA that carries genetic information from the nucleus to the cytoplasm, where it serves as a template for protein synthesis

DNA Footprinting

Gives precise identification of DNA-binding proteins Label DNA molecules at only one end (original DNA molecules are longer and labeled at both ends, but one end is cut off with restriction enzymes, then the two differently-sized fragments are separated with gel electrophoresis before purifying the fragment you want to analyze) Use purified protein to bind to DNA Use nuclease (like DNase-1) or chemical to cleave DNA randomly along molecule, then run fragments w/ gel electrophoresis Large gap on gel indicates "footprint" where DNA molecule had a bound TF protein protecting the sequence from cleavage Sequence fragments (after separating from bound TFs) then compare sequences to determine region bound by TF

Experimental identification of DNA/protein interactions and test function of interaction--does protein bind directly to specific sequence identified? Can I detect protein that binds to sequence of interest?

In vitro: DNAse I footprinting, EMSA; in vivo: ChIP

Provide examples of genetic disorders caused by splicing defects.

Marfan's syndrome = a dominant autosome disease caused by mutations that disrupt splicing of fibrillin gene transcripts (important for walls of heart and vessels). Abnormal splicing of CD44 can cause tumor metastasis. spinal muscular atrophy (SMA) in children ages under 2 - exon 7 gets skipped Altered poly (A) site choice in cancer cells cause shorting of 3'UTRs by alternative cleavage and polyadenylation-->activates oncogenes in cancer cells Mutation in the consensus sequence (AAUAAA) poly A site in globin gene causes thaliasemia

Two classes of aminoacyl-tRNA synthetases

Observed that there were two broad classes of aminoacyl-tRNA synthetases (in bacteria) *Class I* - Attaches amino acids to the *2'OH* on the ribose of the 3'-terminal adenosine - Generally used for the *bulkier amino acids* *Class II* - Attached amino acids to the *3'OH* on the ribose of the 3'-terminal adenosine - Generally used for the *smaller amino acids* Once amino acid is attached to ribose at either the 2' or 3' positions, it isomerizes to the other position, and can be at either position for protein synthesis

DNA Affinity Purification

Purifying a protein when you know DNA binding specificity/sequence Start w/ cell extract of proteins Run extract through column containing *non-specific* DNA sequences--salt wash removes proteins that didn't bind to any DNA Remaining proteins that are shown to have bound to DNA are run through column containing DNA matrix of only the *target binding sequence* for protein being analyzed Successively higher-concentration salt washes purify proteins in column to elute all but the rare protein that specifically recognizes known binding sequence

what are ribonucleoproteins

RNPs a complex with RNA and proteins

tRNA processing

RNase P cuts primary transcript introns that are spliced and ligated all eukaryotic tRNA are modified at the 3' end with the addition of a CCA base modification that can covalently attach amino acid

RNA Seq Tags

Repeated reads from high-throughput sequencing machine corresponding to mRNA transcripts being analyzed Spikes = exons Blanks = spliced out introns that don't make it to the cytoplasm

STAT phosphorylation example

STAT proteins are constitutively expressed in cells that need to respond to immune signals, phosphorylation enables dimerization for nuclear import and then DNA binding

tRNA structure components in order

Starting at the 5' end of the tRNA molecule and going towards 3' end - *5' end base pairs with the 3'end* (3' end has an overhang) - *D arm* (Contains Dihydrouridine) - *Anticodon arm* - *Variable loop* - *T arm* - *Acceptor stem*

wobble position

The first base of the anticodon (which matches the third base of the codon)

describe the steps in pre-mRNA 3' end maturation

The maturation of pre-mRNA occurs via two transesterification reactions; these reaction are not driven by ATP b/c the total phosphodiester bond is conserved Step 1: Attack by the 2' OH of the branch point A. --Branch point A gets activated by snRNA and associated protein.. --Has the 2' -OH which go on a nucleophilic attack b/n exon 2 and exon 1 to break the phosphodiester bond. --liberates an RNA with a lariat structure.. With no 5' end which later gets degraded Step 2. Attack by the 3' OH of exon 1.

Ricin

____ is one of the most powerful toxins known. The protein, isolated from castor bean seeds, depurinates a specific adenine in a conserved sequence of the 28S rRNA, permanently inactivating the ribosome

RF-2

____ recognizes UGA and UAA (pro)

Describe the steps in addition of poly A tail in the maturation of 3' end

The poly A tail stabilizes the RNA, protects 3' end from degradation, enhances translation. and facilitates export of mRNA for translation. 1. cleavage: --endonucleases cleave the RNA to expose the -OH at the 3' end 2. polyadenylation: the poly A tail is (not added by the complementary base of the genome) is added by a different class of RNA polymerase whose job is to just adds A's note: 3' end formation by cleavage-polyadenylation is coupled to termination of transcription by RNA pol II

Translation

The portion of protein synthesis that takes place at ribosomes and that uses the codons in messenger RNA molecules to specify the sequence of amino acids in polypeptide chains

Define RNA splicing

The process of excising introns from RNA and connecting the exons into a continuous mRNA.

Transcription

The process of forming a nucleic acid by using another molecule as a templates; particularly the process of synthesizing RNA by using one strand of a DNA molecule as a template

describe the component of a spliceosome?

The spliceosome is a large ribonucleoprotein complex that assembles on the introns It is composed of the pre-mRNA, over 100 proteins, and 5 small nuclear RNAs (snRNAs)

Aminoacylation reactions

Two step reaction *First step* (amino acid activation) An amino acid (AA) is activated to form enzyme (E)-bound aminoacyl adenylate (1) E + AA + ATP → E∙AA-AMP + PPi *Second step* The amino acid is transferred from the adenylate to tRNA (2) E∙AA-AMP + tRNAAA → E + AA-tRNAAA + AMP Note that the aminoacyl-tRNA link is a high energy bond, which is the source of energy for peptide bond formation on the ribosome

In prokaryotic gene regulation

Transcription and translation occur simultaneously in the cytoplasm, and regulation occurs at the transcriptional level

1. EF-Tu 2. EF-Tu-GTP 3. A site 4. EF-Tu-GDP 5. EF-Ts

Translation Elongation 1. Insertion of aminoacylated tRNA: 1. Aminoacylated tRNAs are bound by __1__ on its acceptor arm. 2. __2__ binds to both the ribosome and charged tRNAs. 3. Proper base pairing of the anticodon with the codon in the __3__ stimulates GTP hydrolysis by EF-Tu. 4. __4__ releases tRNA and ribosome. 5. __5__, the guanine nucleotide exchange factor for EF-Tu, regenerates EF-Tu-GTP for further cycles

eEF-2

Translocation in eukaryotes is similar; ___ is analogous to EF-G

1. EF-G-GTP 2. EF-G 3. EF-G-GDP

Translocation: 1. __1__ binds in the A-site, interacting with both 50S and 30S subunits. 2. GTP hydrolysis results in conformation change of __2__, which causes shift of the entire ribosome. EF-G displaces peptidyl tRNA to P site, along with mRNA. Uncharged tRNA moves to E site and is released. 3. __3__ is released.

Describe step 3 of elongation.

Translocation: P site tRNA leaves ribosome, and tRNA in A site takes its place

eRF-3

____ binding to eRF-1-GTP promotes the latter's hydrolytic activity (euk)

Does the protein regulate transcription? (in vivo)

Use a reporter gene assay: 1) put the reporter gene with promotor containing element in a plasmid 2) put the gene encoding the protein of interest in a plasmid 3) express in cells 4) detect reporter expression; controls: use the same reporter with and without the protein and with and without the same binding site

Now that we know that the protein binds, we want to know--do they regulate transcription? (In vitro)

Use a reporter gene assay: add a template containing promotor element + RNA polymerase complex + NTPs + the protein; detect RNA using a Northern blot or by using radioactive nucleotides instead of normal to directly see the RNA run results on the gel

conformation

When appropriate charged tRNA is bound, appropriate ____ of the ribosome stimulates GTP hydrolysis and releases the elongation factor.

(C2-H2) Zinc Finger

Zinc atom bound by 2 cysteines and 2 histidines Beta strands by cysteines serve as stabilizer Alpha helix by histidines does recognition Ex. 9 Zinc fingers in TFIIIA, binding DNA and RNA Another type is C4 Zinc finger dimers: 4 cysteines around Zinc atom, serves as nuclear receptor

eEF-1α

___ is euk analoge to EF-Tu

Diphtheria toxoid (inactive form of toxin)

___ is the "D" of DPT childhood vaccination.

RF-1

___ recognizes UAG and UAA (pro)

RF-3

___, another GTPase, acts with RF-1 and RF-2 to mediate release of the ribosomal subunits (pro)

Corynebacterium diphtheriae

____ affects the upper respiratory tract, where an inflammatory exudate causes severe obstruction to the breathing pathway, and sometimes suffocation. Forms a fibrous pseudomembrane

ribosome binding site (or Shine-Dalgarno sequence

a sequence in prokaryotes where translation starts when the small ribosome subunit binds to

what is pseudouridine

a uridine base with a N switched for a CH group

Identify on a diagram of a gene the following:

a) transcription start site - marked by the promoter (a +1 or bent arrow symbol) b) introns - should be between 5' GU and 3' AG with an A point in the middle c) 5' splice site - look for GU d) 3' splice site - look for AG e) branch points - an A between the branch points, closer to 3' end f) exons - between the introns, so demarcate those first g) 5' UTR - region between +1 and start codon h) 3' UTR - region after stop codon till end of polyA tail i) initiation codon - 5' AUG j) termination codon - 3' UAG, UAA, or UGA k) polyA site - consensus sequence AAUAAA (or lots of A's)

ATP

activates aa

broadly describe aminoacyl-tRNA synthesis

adapter 1- enzyme with two active sites tRNA adenylated- high energy bond with AMP adapter II- charged tRNA- high energy bond carry amino acid= charged tRNA= aminoacyl-tRNA

step 3 elongation

amino acid in the A site attacks the carbonyl carbon of the P site (catalyzed by peptidyl transferse)

Selenocysteine (Sec, or U)

amino acids are not part of the regular genetic code, but they are coded in the DNA: as stop codons that get modified by other sequences in the mRNA.

EF-Tu

aminoacyl-tRNA binds EF-Tu that binds to the 3' end of amino acid protects the ester linkage, the high energy bond, from hydrolysis and loads RNA into ribosome, contributing to accuracy

describe streptomycin

aminoglycoside bind small ribosomal subunit tuberculosis drug high conc causes bacterial cell death low conc causes slow growth rate

Antiparallel codon-anticodon pairing (example)

anticodon 5'-CAU-3' basepairs with codon 5'-AUG-3', which codes for methionine Anticodon appears to be backwards, but should be read from 5'→3'

open reading frames (ORFs)

areas where there are no stop codons

both

arm on tRNA aminoacyl-tRNA synthetase contacts

aminoacyl-tRNA synthetases

attach the correct amino acid to their tRNA; class I, Phe-tRNA synthetase, and class II, Gln-tRNA synthetase through monomer interaction with anti codon and acceptor stem; discriminate based on specific tRNA bp modifications; direct attachment of an amino acid to a tRNA is not energetically favorable - activate tRNA

aminoacylation

attached aa to tRNA

Transcriptional enhancers

can be found at large distances up or downstream, are useful in cloning, use DNA looping

what are examples of diseases treated by RNA

cancer Cardiovascular disease neurodegenerative diseases

eIF2 control of translation

cells stresses activate a kinase that phosphorylates eIF2 phosphorylated eIF2 blocks transfer of GTP and thus no binding to the charged tRNA

describe adapter II of tRNA synthesis

charged tRNA high energy bond between tRNA- AA

what are small nucleolar RNAs

chemical modification RNA in ribosome biosynthesis non coding RNAs

SILENCERS

cis acting DNA sequences similar to enhancers -binds to transcriptional repressor proteins to turn off transcription

describe the secondary structure of tRNA

cloverleaf D-loop= D base= dihydrouridine anticodon loop- 3 base anticodon comp to mRNA variable loop T PsiC loop- has T-pseudouracil- C acceptor stem- CCA fused to amino acid

multiple regulatory sequences at a given promoter provide

combinatorial control and signal integration (Ex. operons and regulons)

how is RNA degraded so it can be given to the endosome

deadenylase is a exonuclease associated with the 5' and 3' end -starts to degrade 3' polyA tail- gradual -long mRNA- longer in cell as more A's -when there are less than 30 A's decapping occurs -endonuclease cuts mRNA into 2 and is sent to the endosome

list the three steps of elongation

decoding transpeptidation translocation

points of proof reading

decoding step and following GTP hydrolysis by EF-Tu; ribcrosome does not edit

nonsense mediated decay

defective transcripts are removed in eukaryotes due to presence of premature termination codon

what were siRNA's originally for?

defence against dsRNA viruses also for repression and form heterocomplex now we can design artificial siRNA specific forms to control own genes

describe alternative splicing

different forms of protein as some exons removed post transcriptional control depends on where gene expressed can have aminio/carboxy terminal extensions, insertions, deletions, alternative sections

RNA polymerase 2 does/doesnt bind to DNA

does not; binds to proteins bound to DNA

Processing of hnRNA leads to

mature RNA or mRNA

describe postranslocational state

molecular mimicry- secondary elongation factor resembles tRNA so actively displaces tRNA from A site -push to E and P, needed to completely push out -before it binds P/E and A/P sites

IF proteins

molecular switches the block binding in A and E site and dissociate due to GTP hydrolysis and resulting structural changes; help bind initiator met into P site, removal allows large subunit to bind eukaryotic IF2 catalyzes large subunit joining

Transfer RNA (tRNA)

molecules are short RNAs that serve as the adapters between the codons of mRNA and the amino acids they code for.

describe the decoding step of elongation

nucleotide code translated to amino acid code -aminoacyl tRNA binds to A site -elongation factor binded to tRNA- EF-tu= eEFIA -rearranging occurs so right tRNA enters -incorrectly paired tRNAs preferentially dissociate -GTP-> GDP

where can you find the endosome

nucleus to process small RNA (sn and snoRNA) and to degrade if it shouldn't be translated cytoplasm- degrade RNA after a full life

termination

occurs by molecular mimicry; release factors induce peptidyl transfer to water rather than an amino acid that is then released through exit tunnel - prokaryotes - RF will act like EF-G during translocation to remove from A site - eukaryotes - ATPase triggers peptidyl bond transfer to water and release protein and break apart ribosome

amino acid + ATP + tRNA--> aa-tRNA + AMP + Pi

overall equation for aminoacylation

elongation

peptide bond formation occurs via peptidyl transfer reaction that requires a highly conserved rRNA element; the ribosome provides an electrostatic environment that reduces the energetic cost of forming the highly polar transition state by shielding the reaction from bulk water; occurs via nucleophilic attack on the electron deficient center that requires proximity of tRNA conformational change

what is an extensive match with miRNA's

perfect/ close to perfect match occurring in plants arg- does slicing and use ATP to cut into 2 mRNAs rapid degradation by exosome RISC released and recycled- catalytic

How to assess the ability of TFs to change rates of transcription initiation

reporter gene assays-cotransfection where gene is in an expression plasmid and a reporter plasmid contains its binding site plus a reporter gene

TFIIE

required for promoter clearance and elongation (1)

TFIIJ

required for promoter clearance and elongation (2)

what are the directions of translation

ribosome reads in a 5' to 3' direction synthesise N to C terminal, end synthesised first

how do ribozymes cleave RNA

ribozyme does complimentary base pairing to substrate RNA ribozyme complexes with Mg2+ no proteins involves cut target- cleave RNA ribozyme recycled

promote attachment of aa to acceptor stem, strengthen codon-anticodon interaction

role of posttranscriptionally modified bases in tRNA

describe prokaryotic ribosomes and the drugs that effect them

same active centre as eukaryotes 3 rRNA's and less proteins targeted by drugs eg aminogyclosides bind active site to increase error frequency of protein synthesis causing abortions do to bind to human R kanamycin and gentamycin

aminoacyl-tRNA synthetase

selects correct aa for covalent attachment to tRNA

reside within about 150 nucleotides of the RNA start site

sequence specific DNA binding activators

polyribosome, or polysome

several ribosomes following each other down the RNA

Promoter proximal elements

short (~10 NT) sequences 200-40 NT ahead of the promoter that greatly increase transcription rates example-SV40

Eukaryotic initiation scanning

the IF proteins unwind secondary structure and let the initiator tRNA check codons for AUG, may be helped by Kozak sequence

hnRNA is processed by

the RNA polymerase II enzyme and other proteins in 3 consecutive steps during synthesis 1. 5'CAP 2. splicing of introns 3. 3' poly(A) tail

genes are only expressed in cells with __

the correct number of factors, all must be present to cause expression

genetic code

the correspondence between the 64 codons and the amino acid they code for

N-formyl methionine

the first amino acid in the start proteins, In bacteria, the first amino acid is a modified version of methionine

50S subunit

the large subunit of the prokaryote ribosome, has 2 RNAs (one fewer than eukaryotic) plus 31 polypeptides

translocation

the movement of mRNA down 3 nucleotides in the ribosome. Caused by conformation changes by hydrolyzing GTP to GDP.

Translation: Termination

the new polypeptide is released from tRNA due to 2 release factor proteins.

coding sequence or CDS

the part of the messenger RNA sequence that is translated into protein.

reading frame

the same region of DNA can be read in 3 ways, starting one base apart

30S subunit

the small subunit in prokaryotes, has 1 RNA and 21 polypeptides.

40S subunit

the small subunit of a ribosome, contains one RNA plus 33 polypeptides.

Describe how Pre-mRNA Splicing Proceeds Through a Lariat for the 2-hydroxyl group and 3-hydroxyl group

• 2'-hydroxyl on branching nucleotide initiates nucleophilic attack on 5' splice site. • 3'-hydroxyl generated at the 3'-end of the exon during the first transesterification initiates a nucleophilic attack on the phosphodiester bond at the 3'-splice site.

Describe the The 5′ End of Eukaryotic mRNA Is Capped

• A 5′cap is formed by adding a G to the terminal base of the transcript via a 5′-5′ link. • 5' 7-methyl guanosine cap. • The cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation. • Major function is to protect the mRNA from degradation. • Cap is recognised by cap binding protein heterodimer (CBP20/80) to facilitate export from the nucleus. •The capping process takes place during transcription and may be important for release from pausing of transcription. •3 forms of capping. All contain cap 0. The 5′ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated.

Explain why snRNPs Are Required for Splicing

• All the snRNPs except U6 contain a conserved sequence that binds the Sm proteins that are recognized by antibodies (anti-SM) generated in autoimmune disease. • U4 and U6 are found together as a di-snRNP. • Each snRNP is formed in a multistep process.

Explain how Splicing Can Be Regulated by Exonic and Intronic Splicing Enhancers and Silencers

• Alternative splicing is often associated with weak splice sites. • Sequences surrounding alternative exons are often more evolutionarily conserved than sequences flanking constitutive exons. • Specific exonic and intronic sequences can enhance or suppress splice site selection.

Describe An Alternative Spliceosome Uses Different snRNPs to Process the Minor Class of Introns

• An alternative splicing pathway uses another set of snRNPs that comprise the U12 spliceosome. Works in a similar way to major intron splicing (GU-AG). • The target introns are defined by longer consensus sequences at the splice junctions rather than strictly according to the GU-AG or AU-AC rules. • Major and minor spliceosomes share critical protein factors, including SR proteins.

Describe the cleaving of tRNA by endonuclease

• An endonuclease cleaves the tRNA precursors at both ends of the intron. • Release of the intron generates two half-tRNAs with unusual ends that contain 5′ hydroxyl and 2′-3′ cyclic phosphate and a linear intron.

Describe The 3′ End Formation of Histone mRNA Requires U7 snRNA

• Expression of histone mRNAs is replication dependent and regulated during the cell cycle. • Histone mRNAs are not polyadenylated; their 3′ ends are generated by a cleavage reaction that depends on a conserved hairpin structure in the mRNA. • The cleavage reaction requires the SLBP to bind to a stem-loop structure and the U7 snRNA to pair with an adjacent single-stranded region. • The cleavage reaction is catalyzed by a factor shared with the polyadenylation complex.

Describe the characteristics of Autocatalytic Introns

• Found in organelles and in bacteria. Group I and II introns. • Group II introns excise themselves from RNA by an autocatalytic splicing event (autosplicing or self- splicing). • The splice junctions and mechanism of splicing of group II introns are similar to splicing of nuclear introns. • A group II intron folds into a secondary structure that generates a catalytic site resembling the structure of U6-U2-nuclear intron.

Describe the Commitment of Pre-mRNA to the Splicing Pathway

• In cells of multicellular eukaryotes, SR proteins (Splicing regulatory proteins) play an essential role in initiating the formation of the commitment complex. • Pairing splice sites can be accomplished by intron definition or exon definition.

Describe the mRNA Splicing & Export are Coupled Processes- REf1 and EJC

• REF1 (RNA export factor 1) is recruited by a splicing factor (UAP56) and targets mRNA to nuclear pore. • exon junction complex (EJC) - A protein complex that assembles at exon-exon junctions during splicing and assists in RNA transport, localization, and degradation. • mRNA export factors dissociate after export. Factors associated in nonsense mediated decay (NMD) remain bound.

Describe how the Capping enzyme is recruited by the CTD of RNA polymerase II

• RNA pol II CTD must be phosphorylated on Ser-5 to target a transcript for capping . • CE interacts with Ser-5 phosphorylated pol II. •RNA splicing - The process of excising introns from RNA and connecting the exons into a continuous mRNA. •Occurs during and/or after transcription.

Describe Intron and exon definition

• SR proteins interact with one another as well as other proteins to form protein bridges that extend across the intron that is to be excised or across exons. • Exons contain exonic splicing enhancers (ESE) that are binding sites for SR proteins. • When bound, SR proteins recruit U1 snRNPs to the downstream splice site and U2AF to the pyrimidine tract and AG dinucleotide of the upstream 3'-splice site. • U2AF also recruits U2 snRNP to the branch point sequence (A): cross-exon recognition

Explain how Alternative Splicing Is a Rule, Rather Than an Exception, in Multicellular Eukaryotes

• Specific exons or exonic sequences may be excluded or included in the mRNA products by using alternative splicing sites. • Alternative splicing contributes to structural and functional diversity of gene products.

Describe the Nuclear Splice Sites Are Short Sequences in human GU-AG introns

• Splice sites: sequences immediately surrounding the exon-intron boundaries. • The 5′ splice site at the 5′ (left) end of the intron includes the consensus sequence GU. • The 3′ splice site at the 3′ (right) end of the intron includes the consensus sequence AG. • GU-AG or U2-type introns (98% of human introns)

Describe how Pre-mRNA Splicing Proceeds Through a Lariat

• Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of the 3′ splice site. • A lariat is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position at the A at the branch site in the intron. • 2 transesterification reactions. A reaction that breaks and makes chemical bonds in a coordinated transfer so that no energy is required.

Describe how tRNA Splicing Involves Cutting and Rejoining in Separate Reactions

• The 5′-OH end is phosphorylated by a polynucleotide kinase, the cyclic phosphate group is opened by phosphodiesterase to generate a 2′-phosphate terminus and 3′- OH group, exon ends are joined by an RNA ligase, and the 2′-phosphate is removed by a phosphatase.

Describe the The Spliceosome Assembly Pathway

• The commitment complex progresses to pre-spliceosome (the A complex) in the presence of ATP. • Recruitment of U5 and U4/U6 snRNPs converts the A complex to the mature spliceosome (the B1 complex). • The B1 complex is next converted to the B2 complex in which U1 snRNP is released to allow U6 snRNA to interact with the 5′ splice site.

Explain the effect of splicing enhancers

• The effect of splicing enhancers and silencers is mediated by sequence-specific RNA binding proteins, many of which may be developmentally regulated and/or expressed in a tissue-specific manner. • The Nova and Fox families of RNA binding proteins can promote or suppress splice site selection in a context dependent fashion. • The rate of transcription can directly affect the outcome of alternative splicing.

Explain how the 3′ Ends of mRNAs Are Generated by Cleavage and Polyadenylation

• The sequence AAUAAA is a signal for cleavage to generate a 3′ end of mRNA that is polyadenylated. • The reaction requires a protein complex that contains a specificity factor, an endonuclease, and poly(A) polymerase. • The specificity factor and endonuclease cleave RNA downstream of AAUAAA.

Describe the The 3′ Ends of mRNAs Are Generated by Cleavage and Polyadenylation

• The specificity factor and poly(A) polymerase add ~200 A residues processively to the 3′ end. • The poly(A) tail controls mRNA stability and influences translation. • Cytoplasmic polyadenylation plays a role in Xenopus embryonic development.

Describe how The 3′ mRNA End Processing Is Critical for Termination of Transcription

• There are various ways to end transcription by different RNA polymerases. • The mRNA 3′ end formation signals termination of Pol II transcription. • The Elongation complex changes conformation upon recognising the poly(A) site. • After cleavage a splicing factor (SF) recruits Xrn2 which digests downstream RNA until it reaches RNA polII. • PolII is released with help of a helicase?

Describe how The 3′ mRNA End Processing Is Critical for Termination of Transcription in pol I and pol III

• Transcription by Pol I and Pol III uses specific terminators to end transcription. • Pol I: 2 discrete termination sites are recognised by a DNA-binding protein (TTF1 in mouse) and cleavage mediated by the endonuclease Rnt1. • Pol III: Defined terminator sequence in the DNA molecule (oligo dT) signals release of RNA polymerase.

Describing The splicing reaction proceeds through discrete stages.

• When U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytic active site. • Both transesterification reactions take place in the activated spliceosome (the C complex). • The splicing reaction is reversible at all steps.

Describe the Mutations in splice sites.

• exons are recognized as units • mutation in splice sites (ESE) can result in skipping of entire exon or insertion of wrong sequence • Growth Hormone Deficiency Type II - mutation in splice site induce exon skipping • BRCA1 in some of breast and ovarian cancers, mutation destroys an acceptor site leading to a 59 base-pair insertion

Explain why snRNAs Are Required for Splicing

• small cytoplasmic RNAs (scRNA; scyrps) - RNAs that are present in the cytoplasm (and sometimes are also found in the nucleus). • small nuclear RNA (snRNA; snurps) - One of many small RNA species confined to the nucleus; several of them are involved in splicing or other RNA processing reactions. snRNAs exist as ribonucleoprotein particles (snRNA + several proteins) = snRNP or snurp. • small nucleolar RNA (snoRNA) - A small nuclear RNA that is localized in the nucleolus. • Each snRNA is present in its own small ribonucleoprotein particle. • The five snRNPs involved in splicing are U1, U2, U5, U4, and U6. • Together with some additional proteins, the snRNPs form the spliceosome. • The spliceosome is ~12 MDa. Five snRNPs account for almost half of the mass. • splicing factor - A protein component of the spliceosome that is not part of one of the snRNPs. • Complex assembles sequentially on the pre-mRNA and passes through several "pre-splicing complexes" before forming the final, active complex = spliceosome.

Explain how tRNA Splicing Involves Cutting and Rejoining in Separate Reactions

• tRNA splicing occurs by successive cleavage and ligation reactions. • RNA polymerase III terminates transcription in a poly(U)4 sequence embedded in a GC-rich sequence. • All tRNA introns include a sequence that is complimentary to the anticodon of the tRNA. This creates an alternative conformation for the tRNA arm. • The intron in yeast tRNAPhe base pairs with the anticodon to change the structure of the anticodon arm.

Explain how Alternative splicing of the CaMKIIδ is different

•Alternative splicing of the CaMKIIδ gene: 3 different alternative exons target the kinase to different cellular compartments.

Explain how RNA is modified in the nucleus

•RNA is modified in the nucleus by additions to the 5' and 3' ends. •Poly(A) modification is intrinsically link to transcriptional termination. •mRNA is exported.

Describe the Nuclear Splice Sites Are Short Sequences in human AU-AC introns

•The 5′ splice site at the 5′ (left) end of the intron includes the consensus sequence AU. •The 3′ splice site at the 3′ (right) end of the intron includes the consensus sequence AC. •AU-AC or U12-type introns (0.1% of human introns)