BMSC 220
homeodomain proteins
(contain a helix-turn-helix DNA binding domain) play critical roles in the regulation of gene expression during embryonic development.
Nucleosome core particle
147 base pairs of DNA wrapped around octamer consisting of 2X(H2A,H2B,H3,H4)histones
Chromatosome
166 BP with a H1 histone (includes the nucleosome structure
Disaccharides
2 monosacharides together such as sacrose and lactose
Purines
A, G
Pyrymadines
C, T, U
Carboxyl group
COO-group on an amino acid
Chromosome
Carriers of genes consisting of long DNA molecules and associated proteins number of chromasomes not related to complexity
RNA polymerase
Enzymes that transcribe RNA from DNA template
• Amino acid structure
H3N - CHR-OO
Glycosidic Bonds
Link monosacharides together
translation
Process of synthesizing protein from mRNA template
co-translational Step 3:
Ribosome sits over translocon channel and polypeptide chain is protein synthesized into the channel
single-stranded DNA-binding proteins,
Stabilizes the unwound single stranded template DNA
role of 5 and 6 carbon sugars in the cell
Storage, energy and building block for other molecules (DNA/RNA)
Alternative splicing
The generation of different mRNas by variation in the pattern pf pre-mRNa splicing may explain increased diversity of proteins
3' hydroxyl group
The hydroxyl group on the 3' carbon of the sugar. Will form bond with phosphate group
discuss what defines the structural and functional characteristics of a given protein
The interaction between elements defines the structure and the structure (form) defines the function. Lock and key - if it doesn't fit it wont work
Why must membrane proteins be inserted into the ER membrane into their correct orientation before they are moved to another lipid bilayer?
They cannot be flipped within the membrane
- What do all extracellular matrices have in common?
Tough fibrous structural proteins embedded in gel like polysachatides (groud substance) Adhesion proteins anchor cells in ECM
What is the difference between transcription and translation?
Transcription creates an RNA copy from a DNA template in the nucleus Translation is carried out on ribosomes with tRNAs serving as adapeters between the mRNA template and the amino acids being joined together
RNA Polymerase III
Transfer RNA
- assembly
Tubulin polymerizes (1 alpha and 1 beta isoform) to form dimer, form 13 linear protofilaments
Phospholipids
Two fatty acids attached to a polar head
RNA polymerase I
Used for rRNA in 5.8s, 18S, 28S
Gene
a segment of DNA within a chromosome that is expressed to yield a functional product
Describe how mannose-6-phosphate residues target proteins to lysosomes
additionof the mannose-g-phosphate residue provides a binding place for M6P receptors associated with clathrin. Clatherin coated vesicles sort to the lysosomes
What are the two types of stable adhesion junctions called? To what sort of INTRAcellular structures does each bind to/ connect between cells?
adheren junction and desmosomes adheren - actin to cytoskeleton desmosome - epithelial cells - intermediate filament
how transcription factors can be isolated by DNA-affinity chromatography
agrose beads to stick to specific factor to isolate it
tRNA
align amino acids in the mRNA template
snRNA snRNPs
assist in targeting snRNPS to splice junctions through hybridization to RNA transcript -are catalytic and carry out steps in the splicing reaction small nuclear riboonucleoprotein complexes -complexes of snRNAs that play central role in splicing process
What is a barbed end? Pointed end? Why does it matter? How does GTP/ GDP play into this?
barbed end adds monomers fadster than pointed end ATP actin associates with chain more readily than ADP actin ADP actin disassociates more easily
Nucleoside
base with sugar
Nucleotide
base with sugar and phosphate group
nucleosome
basic structural units of chromatin DNA + Histone
Why is it critical that lysosomal proteins are only targeted to lysosomes?
because most of the enzymes destined for the lysosomes are only active in the acidic pH of lysosomes ; and therefore will not work that well in the more neutral pH of the cytoplasm; 2) even though this is the case, you don't want these enzymes even being minimally active or available in the cytoplasm because they will destroy almost everything in their path)
• Describe the structure of the plasma membrane as a semi-permeable barrier.
bilayer of amphipathic phospholipids with associated proteins and specialized lipids • lipids 50% of mass of membrane • Separate two aqueous compartments • uncharged/hydrophobic molecules can diffuse while charged molecules require transport proteins
Point #4: Discuss (briefly) how promoters and enhancers regulate the rate of transcription by RNA polymerase II.
bind to promoter and enhancer sequences. Alter the efficiency of the pre initiation complex
Phosphodiester bond
bond between the 5' phosphate group and the 3' hydroxyl group
Starch
branched or unbranched α glucose polymer Storage in plants
Glycogen
branched α glucose polymer - Storage in animal cells
12. The structures and/or organelles associated with the secretory pathway are the: a. plasma membrane, vesicles, mitochondria, lysosomes, RER. b. Golgi apparatus, mitochondria, lysosomes, endosomes, plasma membrane. c. RER, Golgi, lysosomes, vesicles, plasma membrane. d. intermediate filaments, lysosomes, Golgi apparatus, plasma membrane, SER.
c
topoisomerases
catalysezes the breakage and rejoining of DNA strands to relieve torsion caused by unwinding DNA
Define euchromatin
decondensed - transcriptionally active interphase chromatin
define "enhancer sequence" and describe how it influences transcription
either upstream or downstream gene specific bind TFs
telomerase
enzyme that maintains the ends of eukaryotic chromosomes has reverse transcriptase capability
list and define the DNA sequences that are downstream from the start of transcription (exons, introns, stop)
exons - transcribed DNA introns - non transcribed DNA Stop sequence - tells RNA Pol II to stop
What is G-actin/ F-actin?
f - filamentous formed of head to tail monomers of G actin
- The pre-initiation complex (initiation of transcription by RNA pol II):
forms in stepwise manner - initiation of transcription - does not modify rate of transcription initiation - necessary but not sufficient for transcription
- basal transcription complex
forms in stepwise manner - initiation of transcription - does not modify rate of transcription initiation - necessary but not sufficient for transcription
- transcription initiation complex
forms in stepwise manner - initiation of transcription - does not modify rate of transcription initiation - necessary but not sufficient for transcription
Which types of molecules can vs. can't freely diffuse through the plasma membrane
gases hydrophobic molecules small polar molecules cant large polar molecules charged molecules
upstream promoter/ Enhancer
gene specific transcription factors bind to upstream promoters and enhancers control the rate of transcription
genetic code
information from nucleotide triplets that encode for amino acids in proteins
Where is the codon? The anticodon? When is the tRNAmet brought into the whole mix? (see notes on step 1)
initially both are at the P site The codon is on the mRNA the anticodon is on the tRNA base subsequent pairings are at the A site
What are the three general steps of translation?
initiation elongation termination
What are cadherins?
integral plasma membrane proteins that link the cytoskeleton intracellilarly with each other extracellularly
Describe the proteins in the plasma membrane which bind to both the extracellular matrix structures and to the cytoskeleton, and the two types of structures these complexes form
integrins
• hydrophilic
interact well with water • tend to interact on the surface of proteins
What is the "genetic code"?
is a triplet code.(each triplet of nucleotides in mRNA is a codon that specifies insertion of a specific amino acid) every three nucleotides is a codon that specifies a specific amino acid
Define amino acid
linear chains of amino acids
- How are intermediate filaments involved in cell-EMC interactions (hemidesmosomes) and cell-cell interactions (desmosomes)
linked to hemidesmosomes and desmosomes
role of lipids vs. proteins in the structure of the plasma membrane
lipids provide structure and proteins function. 50/50 by weight 1:50(100) protein to lipid in number
Amino acid side chain groups
one of 20 side chains
Discuss4 reasons why gene regulation is crucial to cell function
one of the fundamental ways to modify activity in response to 1. environment 2. cell cycle 3. distinct cell activity 4. cell differentiation in development
Define Proteins:
one or more series of amino acids
What is clathrin?
one type of vesicle protein coat that has reptors for M6P and causes vesicle to be sorted to lysosomes
Base paring
pairing between A -T (U) and C-G
• amphipathic
part soluble part insoluble
Amphipathic
part water soluble part insoluble
endocytosis
process of material being internalized by a cell
- structure & composition of micro tubules
rigid hollow rods. polar Alpha end - + end - fast growing beta end - neg end - slow growing
• Steroid
rings • polar hydroxyl group • pass through membrane to the nucleus
nuclear envelope
two phospholipid by layer
, RNAseH,
- Enzyme that degrades RNA in eukaryotes
Polysaccharides
100 to thousand such as glycogen or starch
TBP
TATA Binxing Protein
inherited traits
specifies a specific traite. Many different allees for any one gene
sliding clamp proteins
- load and hold polymerase onto template
How many subunits are there on ribosomes? What are they generally called?
2 sub units euk 60S and 40S proc 50S and 30S
List the major similarities and differences between prokaryotic and eukaryotic cells; animal vs. plant cells
Characteristic, prokaryote, Eukaryote Nucleus, no, yes Diameter, 1picometre, 10-100 picometer Cytoskeleton, No, Yes Organells, no, yes DNA content, few, lots Chromosomes, single circular DNA, multiple linear DNA
Describe the basic characteristics (in terms of charge/ polarity, abilities to form bonds, etc.) of lipids
Fatty Acids have polar head and non polar tail Tri glyceriroles (fats) 3 fatty acids linked to a glycerol molecule
What is the role of ERGIC in the secretory pathway?
Is an intermediate with mixed propertieof the ER and Golgi
Describe the basic characteristics (in terms of charge/ polarity, abilities to form bonds, etc.) Carbohydrates
Non polar Has ability to form glycosidic bonds
co-translational Step 2:
SRP binds to its receptor on ER/ translocon (the receptor on the ER where the ribosome will sit during translation)
TFIIH plays the final role in transcriptional activation (phosphorylation and helicase)
The C terminal on TNA pol II is phosphorylated. Helicase breaks H bond in dsDNS Mediator and associated preinitiatioon comples proteins release from RNA pol II
What is the general function of the Golgi? Why do most (if not all) proteins destined for the ER travel to the Gogli and then back again to the ER? Where does the actual sorting of proteins therefore start?
The golgi processes and sorts proteins. Processing includes the synthesis and/or modification of the carbohydrate portion of the glycoproteins Soriting starts in the Golgi
What is myosin? What does it do?
a protein that interacts with actin and acts as a molecular motor. Uses ATP
proximal and distal promoter elements
binding sites of gene specific transcription factors that modulate activity of basal transcription complex. Change rate of transcription initiation or rate of basal transcription complex assembly can be either activators or repressors
How do matrix adhesion proteins link the EMC to itself and to the cell? Where are they located? How many binding sites do they have?
both laminin and fibronectin have 3 binding sites They bind to collagen, proteoglycans and to the cell
Describe the basic structure and functions of the matrix polysaccharides which are located in the extracellular matrix
bottle brush structures that give bulk to ECM and soak up water
7. A primary culture of mammalian liver cells is capable of only 50-80 normal cell divisions before they suddenly die due to instability and degradation of the chromosomes. What is the most likely reason for this observation? a. a. lack of histone deacetylase (HDAC) activity in the cultured cells b. b. the origins of replication are no longer active in the cultured cells c. c. a lack of telomerase activity in the cultured cells d. d. the cultured cells exhaust their supply of DNA polymerase after multiple cell divisions e. e. the cells could no longer play well together
c
rRNA
component of ribosomes
• hydrophobic
does not interact well with water • tends to be inside a protein • tends to be within the cell membrane for transmembrane proteins.
karyopherins / importins
example of nuclear transport receptor
snRNP
form splisosomes to remove introns and splice exons together
, heterochromatin
highly condensed transcriptionally inactive chromatin
how do structures such as tight junctions polarize the cellular distribution of proteins?
prevent the ingress of molecules between cells and separate apical and basal domains
, Lagging strand,
synthesis in a iscontinuous manner replication opposite direction of fork movement
Describe the basic structure of the plasma membrane in terms of:
...
• 4 catagories of cellular organic molecules
Carbohydrates • lipids • nucleic acid • protein
Discuss the signaling functions of nucleotides
Cyclic GMP
reverse transcriptase
DNA polymerase that uses RNA template
Reaction to form glycosidic bonds
Dehydration
What are glycosaminoglycans? How do they help create a gel-like matrix?
GAGs Gel forming polysaccharides negative charge attracts and traps water
Collagen
Major structural proteinint eh intercellular matrix
BiP
Molecular chaperon in the ER
- discuss the role of polar vs. non polar parts of lipids (espec. phospholipids)
Phospholips cell membranes Non polar
What similar types of functions does each cytoskeleton group have?
cell shape, structural support,
proteolysis
degradation of ubiquitin marked proteins by proteasomes
RNA splicing
joining of exons in a precursor mRNA molecule
• - Give some examples of types of intermediate filaments / types of cells they are in (* on Slide 11).
keratins - wpithelial cells • neurofilament proteins - mature nurons • nuclear lamins - nuclear lamina of nucleus
ced 9
negative regulator of apoptosis cells with mutaion die
What is co-translational transport and how does it occur?
transport of the active ribosome complex to the ER. A signal near the leading N terminus is SRP bind to signal SRP receptors on ER bind to SRPs Tranlocon
• Phospholipid structure
two fatty acids joined to a polar head • amphipathic (part soluable part insoluable
it being a "phospholipid bilayer"
two layers of phospholipids forms a a stable barrier between two aqueous compartments
Cellulose
unbranched \beta glucose - cell walls
Discuss which molecules can readily diffuse across phospholipid bilayer, and which require specific transport systems
uncharged/hydrophobic molecules can diffuse while charged molecules require transport proteins
- What is the common structure of an intermediate filament? Does it have polarily?
1. central alpha helical rod 2. N and C terminus change the function 3. phosphorylation makes unstable 4. no polarity
Oligosaccharides
3 or more monosacharides
Nucleoside
Base and Sugar
Nucleotide
Base sugar and phosphate group
1. The eukaryotic small ribosomal subunit initially binds at what structure on the mRNA? a. A Shine-Dalgarno sequence b. The poly A tail c. The 7-methylguanosine cap d. A TATA sequence e. An AUG initiation codon
C
11. The signal recognition particle (SRP) a. recognizes the signal sequence of newly transcribed mRNAs and targets them to the ribosome. b. recognizes the signal sequence on the amino terminal of proteins and targets them to the ribosome. c. recognizes the signal sequence on the amino terminal of proteins and targets them to the RER. d. recognizes the signal sequence on the amino terminal of proteins and targets them for cleavage by the signal peptidase. e. recognize the signal sequence of tRNAs and targets them to the signal processing centre of the ribosome.
C
- Lipid functions
Cellular membranes, more efficient storage, hormones
main functions of the nucleus
Central role in gene expression and genome activity • repository for genetic information • DNA replication • RNA transcription • RNA processing • Ribosome assembly
BiP
Chaperon found in the lumen of ER
Describe the differences between gene regulation between prokaryotes vs. eukaryotes
Characteristic/ Euk/ Pro Chromatin / Yes / no (free DNA Compartmentalization / Yes / No Extensive RNA processing / Yes / No Multiple RNA polymerase / Yes / No (only one)
Where is the cis vs. the trans face of the Golgi with respect to the nucleus?
Cis closest to ER (where proteins enter) trans furthest from ER (where proteins exist)
What protein is the primary structural component of the ECM? What sort of structure does it form? How is it modified to increase its' hardness?
Collagen is the major structural protein. Form straight fibrils and fibres triple helices configuration
list and define the DNA sequences that are upstream from the start of transcription (Inr, TATA box, proximal promoter sequences) and describe the role each plays in transcription
Core promoter region with INR and TATA- yes/no switch Upstram promoter region for gene specific TFs - Dimmer switch
1. general function (what are the two broad general functions of the plasma membrane?)
Determines the composition of the cytoplasm 2. mediates interaction between cell and outside environment/cells
2. In the process of translation: A) a strand of mRNA is formed with nucleotide sequences complementary to those of DNA B) nucleotide sequences of tRNA are established C) a polypeptide is formed in response to the rRNA nucleotide sequence D) rRNA is synthesized with sequences complementary to those of tRNA E) a polypeptide is formed as dictated by the nucleotide sequence in mRNA
E
• types of lipids
Fatty acids - Simplest lipids • Triacylglycerols - store fatty acids • Phospholipids - component of cell membrane • Glycolipids - in cell membranes • Cholestrol - in cell membranes • Steroid hormones - signaling molecules - estrogen, testosterone, cholesterol
What are proteoglycans? What sort of structure does it look like?
Gags covalently linked proteins large branching structures
- List the major tissue systems of plants (3),
Ground tissue - dermal tissue - vascular tissue
HDAC
Histone Deacetylase - makes histone non-permissive
HAT
Histone acetylase - makes histone permissive
how chromatin is remodeled by HATs and HDACs to enhance/ inhibit transcription
Histone must have a reduced charge to allow DNA freedom. Histone tal is acetylated by HAT reducing the charge and deacetylated by HDAC re introducing the charge
Review the general means by which a mature mRNA sequence is translated to a protein
Information transfer from gene to protein can be controlled at the level of translation, and this control is an important element of gene regulation in both prokaryotic and eukaryotic cells.
Discuss how molecules are (or are not) free to move in the lipid bilayer
Molecules are free to spin or move in lateral directions. cannot flip sides without Flipase and energy
Discuss the issue of genome complexity vs. phenotypic complexity in the evolution of multicellularity
Much DNA for cellular functions is the same phenotypic complexity is not generated based on increased genetic complexity. Alternative splicing or other possibilities may account for complexity
Where is the N vs. C terminus of a polypeptide in terms of which part is first synthesized?
N terminus is synthesized first
Describe the role of particular nucleotide sequences in the genome (ori; ARS), and the protein complexes which bind these sequences (ORC)
ORI is the point
Objective #6: Discuss the primary steps involved in the conversion of a pre-mRNA to mature mRNA in terms of why each step is performed (i.e., the function of each step), and discuss the basic mechanics of each step for:
Occures in the nucleus
Caspases
Proteases that effect cell death. Remain domant until required
Transport across nuclear envelope
Small hydrophobic - free small polar - passve larger polar - active transport Nuclear localization signals (in proteins identified by nuclear transport receptors
histone
Small protein with ARG and LYS that facilitate binding DNA sugar phosphate backbone H1,H2A,H2B,H3,H4
the Mediator protein influence transcription
The mediator is a protein complex that links the general factors to the gene specific transcription factors
How are focal adhesions involved in cell movement? (a more difficult question).
actin growth and branching on leading edge actin contracting at trailing edge
How can actin filaments be stabilized/ destabilized? What does ARP do and where does it do this?
actin is stabilized with a capping protein ARP branches the actin chain
What are the difference between actin networks and filaments; and what proteins to they involve? What types of structures would you see them in?
actin networks - cross linked in orthogonal arrays to form 3d meshwork - cytoskeletal framework- formed with proteins Actin bundles - cross linked into closely packed arrays - microvilli in intestinal epithelia - formed with protein
ced 3, ced 4, ced 9
apoptosis genes. code for caspases
What are aqueporins? Ion channels? What is a gated channel?
aqueporins - water channels ion channels - allow movement of select ions. can be gated and open only in response to signaling molecules
Define: 5'UTR, coding sequence, 3'UTR;
area at the 5' end and 3' end that are not transcribed
What is the decoding center?
area within the small ribosomal subunit that ensures proper pairing
in RBCs, what is the contribution of actin, spectrin, and ankyrin in the movement of transmembrane proteins?
association with membrane proteins determines cell shape
ARS
autonomously replicating sequence origin of DNA replication in yeast
9. Which of the following mutations in a gene encoding a protein that is normally destined for secretion from a cell would likely cause that protein to instead remain in the cytoplasm? a. mutation of the gene such that the final 30 amino acids (at the C-terminal of the protein) are missing from the protein b. mutation of the gene such that polyadenylation of the mRNA does not occur c. mutation of the gene such that the first 30 amino acids (at the N-terminal of the protein) are missing from the protein d. mutation of the gene such that the SRP encoding portion of the mRNA is not expressed
c
1. what controls protein mobility within the plasma membrane? (3 main factors)
cant change between inner out layer 2. cytoskeleton anchores 3. separation of basolatral and apical domains
Briefly describe what is meant by carrier and channel proteins. How does the glucose transported work?
carrier -bind specific molecules to be transported channel - open pores through membrane allowing free diffusion Glucose transporter changes conformation when in transporter allowing movement across cell membrane
Translocon
channel into ER for polypeptide chains with signal sequence
Estrogen and testosterone are derivatives of and act as
cholesterol signaling membranes
pre-tRNA processing
cleaved from transcript addition of nucleotides to 3' end modification of bases
What are Gap Junctions?
communication bridge allows ions to pass
5' end 7-methylguanosine cap addition
completed before the end of transcription increases the stability of mRNA
Telomeres
consist of a simple sequence of DNA that maintains the end of linear chomosomes
• Describe the basic characteristics (in terms of charge/ polarity, abilities to form bonds, etc.) of Nucleic Acids
consist of nucleotides (purines, Pyrimidine, phosphorylated sugars • form phosphodiester bonds between 5'phosphate and 3' hydroxyl group • form poly nuceotides • form complementary pairing between NA with hydrogen bonds • Nucleotides paly role in energy ATP and signaling cyclic GMP/AMP
signal peptidase
cuts off the signal portion of the peptide chain from the translocon
- role in mitosis and the centrosome (How was the dynamic instability of microtubules shown with drugs? What kinds of drugs are used in cancer treatment and how do they work? What is the role of microtubule polarity in mitosis?)
determin cell shape and are involved in cell movement locomotion, intracellular transport of organelles, separation of chromosomes during mitosis
DNA ligase
enzyme that seals breaks in DNA strands
mediator
large protein complex that binds to the preinitiation complex and plays a key role in linking the general transcription factors to the gene-specific transcription factors that regulate gene expression
Types of RNA
mRNA - Messenger rRNA - Ribosomal tRNA - Transfer
RNA polymerase II
mRNA less than 10% of total
codon
nucleotide triplet basic unit of the genetic code
Cell membranes contain
phospholipids, glycolipids, cholesterol
of post-translational modifications of proteins including:
phosphorylation - activation, glycosylation - glycoproteins lipid addition - lipoprotein
Active transport
requires energy
Where/ what structures does the secretory pathway involve?
ribosome, Endoplasmic reticulum, golgi apparatus, lysosomes and associated vesicles form part of the secretory pathway
nucleolus
site of ribosome assembly rRNA
Steroid hormone receptors
(contain a zinc finger domain) are transcription factors that regulate gene transcription in response to hormones such as estrogen and testosterone.
Define the term "cell"
, two, The cell is the smallest unit that can perform all life processes.
pre-rRNA processing
- 3 from RNA pol 1 are all product of one gene repeated in multiple tandum copies -pre rRNA is transcribed simultaneously in many areas. - all three rRNAs on pre rRNA -pre eRNA is cleavedS,to form individual ribosomal RNAs (28S,18S,5.8S)
Discuss the pros and cons of the model organisms used in the study of cells: 1. Bacteria/ E. Coli 2. Yeast 3. C. elegans 4. Drosophila melanogaster (fruit fly) 5. Xenopus laevis (zebrafish) 6. Mice
- E.coli - simple, devide quickly, small genome, biosynthetic reactions, DNA/gene cloning/manipulation - yeast - small genome, grow easily/quickly, same approaches as E.Coli, yeast mutants, DNA replication,trans , RNA processing, contaminates lab - C.Elegans - simple multicellular, cell lineage known, easy growth, cheap - Fruit fly - easy to keep/breed, development pattern formation, segmentation - zebrafish - study egg development, easy to keep, transparent embryos - mice - easy to keep, complete genome sequence
List the major tissue systems animals (4-5); and (for animals) their general functions and properties.
- Epithelial - protection secretion absorption, body and organs - connective - bone, cartilage, adipose tissue fibroblasts(fill space between organs and tissues - Blood - Red/white blood cells - Nervous - nerve cells and supporting cells and neurons - Muscle - Voluntary(skeletal), involuntary(smooth), spontaneous (cardiac)
Slides 13-15: Discuss how molecules are transported through the nuclear membrane out into the cytoplasm, and what types of molecules are transported out of the nucleus.
- NES signals target proteins for export - interact with exportins - most RNAs are transported as ribonucleoprotein complexes
relationship between the inner and out membranes of the nucleus and other cellular organelles
- Outer membrane continuous with ER and bond to the cytoskeleton - inner membrane bind to the nuclear lamina
What are the three steps of tRNA processing?
- RNA cleavage - covalent addition of CCA at 3' end - Base modification
List the three nuclear eukaryotic RNA polymerases; their functions (types of RNAs synthesized), and their key characteristics:
- RNA polymerase I: rRNA: 5.8S, 18S, 28S - polymerase II: mRNA: messenger RNA - RNA polymerase III:tRNA(also 5S-RNA)
SR splicing factors
- Serine-Arganine rixh splicing factors - bind to exon sequence to direct splicesome - assist in determining the "choice" of exons that will appear in any one cell type for alternatively spliced mRNA
What are the three steps involved in the "charging" of a tRNA?
- activation of amino acid (ATP) - addition of amino to tRNA - charged tRNA (aminoacyl tRNA) each aminoacyl recoginizes 1) the correct amino acid; 2) the correct codon on the mRNA.
transcriptional activators and repressors work
- blocking binding of transcriptional activators to DNA sequence elements -repressing mediator activity -interaction with co-repressors to modify chromatin structure
Microscopy: light (bright-field), phase contrast/ differential interference-contrast, fluorescence, scanning Electron microscopy, transmission electron microscopy
- bright field - requires fixation - phase contrast - allows visualization of live cells, outside only, small samples only - Fluoresence - uses markers and dyes (GFP) used to see protein/structures in living cells -transmission electron - thin samples -Scanning em - outside of coated samples.
What are focal adhesions and hemidesmosomes? What is their function? Where do you find these cell- extracellular interactions?
- cell matrix juntio that attaches cells to ECM - link to actin filiments - epithelial cell attachments - integrin to laminin - link to intermediate filaments
Describe the role of accessory proteins which act at the replication fork to facilitate DNA replication:
- clamp loading protein helps load sliding clamp protein on each strand - clamp loading protein detaches -Slidding clamp loads and holds DNA polymerase -helicase unwinds DNA ahead of replication fork -single strand DNA binding proteins keep single strands from reattaching between helicase and polymerase -topoimerases - break and rejoin DNA strand ahead of the fork to relieve tension caused by unwinding
exonuclease
- enzyme activity that hydrolyzes DNA or RNA - performed by DNA Polymerase in prokaryotes -RNAseH
helicases,
- enzyme that catalyse the unwinding of parental DNA (uses ATP)
Describe the ends of chromosomes are replicated (telomerase), and how the accuracy of replication is ensured during DNA replication (fidelity, proofreading) and maximized:
- leading strand overhangs lagging strand -telomerase binds with RNA strand - telomerase reverse transcriptase activity builds leading strand - telomerase detaches and lagging strand is extended by primase, polymerase -RNA primer removed
clamp loading proteins,
- loads clamp proteins onto replication fork at primer
What are the first two controls for the specificity of amino acid incorporation into a protein?
- matching correct amino acid to specific tRNA (specificity of enzyme) - correct base paiting of codon and anticodon
Nuclear transport receptors
- mediate transport across the nuclear envelop for NLS equipped proteins
Describe the "polar" nature of actin
- negative end (pointed end) slow monomer addition - positive end (hooked end) rapid momomer addition
splicing
- performed by snRNA and Spliceosomes -5' end of an intron is cleaved and attached near its 3'end (AG link) -intron is cleaved at 3' end -exons are joined
what are the major types of membrane proteins and where are they located?
- peripheral - not inserted into the hydrophobic bylayer - integral - inserted or transmembrane proteins
Describe how the two different (antiparallel) strands of parental DNA are replicated, and define:
- primase adds RNA primer to lagging strand - DNA Polymerase elegates the primer strand - RNA primer degraded by exonuclease activity - gaps in Akazaki fragments joined by DNA ligase enzyme
3' end poly A tail
- recognizes stop sequence and cleaves sequence. - adds sequence of 200 adenine - added to 3' by poly A polymerase -protects the mRNA molecule from enzymatic action in cytoplasm -helps export mRNA from nucleus
Discuss the properties of water which are critical for the structure and function of a cell in terms of its: - Solubility - Polarity Hydrogen bonding abilities
- solubility is critical in many cellular processes - Polarity of water allows hydrogen bonding of water to itself and other charged molecules
What are intergrins? Where are they located? How many different bind domains do they have, and where is each domain located? How do they link the ECM to the cytoskeleton?
- the major cell surface protein attaching the cell to ECM - two domains one in cell oe ECM - bind to ECM and cytoskeleton
Biochemical: subcellular fractionation
- uses centrifugation - different subcellular components fall out at different speeds.
the ionic gradient of a typical mammalian cell
-60mv less sodium inside
Discuss the role of centromeres, kinetochores, and telomeres in mitosis and their basic structure/ composition
-Centromeres are where the sister chromatine join together, -kinetochore protein is used in mitosis and meiosis telomeres are caps on the end of chromosomes to provide stabilization
ori
-Origin of replication -DNA sequence that is the binding site of the protein complex that initiates replication - one or many (more complicated more origins)
ORC
-Origin recognition comples - recognizes core ARS sequence and binds to it to form replication bubble
Replication, Transcription, Translation
-Replication is the synthesis of a duplicate copy of a DNA molecule (DNA polymerase) -Transcription is the synthesis of an RNA molecule from a DNA template (RNA polymerase) -Translation is the synthesis of a polypeptide chain from an mRNA template (ribosomes)
Describe the basic structure of ribosomes in prokaryotes and eukaryotes
-rRNAs are components of ribosomes -complex secondary structures -numerous proteins to form a subunit -similar but different between pro and euk
How is proofreading accomplished?
-selective removal of mismatch bases by DNA polymerase - if mismatch DNA polymerase removes mismatch with 3'-5' exonuclease
4. Describe the role of microtubules in the cytoskeleton in terms of their:
...
Briefly describe how the extracellular matrix differs in different types of body tissues (i.e., between epithelial vs. connective tissue)
...
Briefly describe how transmembrane proteins are processed during translation in the endoplasmic reticulum
...
Cellular organelles involves in each pathway
...
Cytosolic vs. secretory pathways (branches)
...
DNA packaging
...
DNA polymerase requires a primer and cannot initiate synthesis de novo. What serves as a primer for DNA replication? a. A protein with a free OH group b. Short fragments of DNA complementary to the template strand c. The DNA forms a loop resulting in the formation of double-stranded hairpins at the end of the DNA molecule, and these hairpins serve as primers. d. Short fragments of RNA complementary to the template strand
...
Describe how substances are carried by endocytosis to and from the plasma membrane
...
Describe how the different structure of mRNA in prokaryotes vs. eukaryotes dictates differences in how translation is initiated; and some of the general differences between eukaryotic vs. prokaryotic translation
...
Describe the basic organizational structure of the eukaryotic genome:
...
Describe the basic structure and function of matrix structural proteins:
...
Describe the basic structure and functions of the primary matrix adhesion proteins
...
Describe the role of intermediate filaments in the cytoskeleton in terms of their:
...
Discuss how chromatin is regulated to control the transcriptional availability of genes
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Discuss passive and facilitated diffusion across the plasma membrane with respect to:
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Discuss the mechanisms by which molecules are transported through the plasma membrane by active transport in terms of:
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In order to finish your degree, you must figure out the quickest and least expensive way to determine if a new experimental drug accelerates the development of the eye lens. Which of the following would be the best thing for you to do? a. panic b. order some zebrafish in which to do your experiments c. grow an eye lens in culture and administer your drug to the culture d. order a transgenic animal with a mutation known to decelerate lens development and test your drug in the animals
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Objective #5: Discuss how transcription factors and chromatin structure regulate transcription:
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Outline the basic steps involved in the elongation of the polypeptide chain during translation in eukaryotes
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Point #2: Discuss where the key proteins which play a role in transcription (i.e., transcription factors and RNA polymerase II) bind on the gene to be transcribed in terms of the:
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Point #5 (the rest of the slides in this objective): Discuss (briefly) how:
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The twisting of the parental DNA strands around each other ahead of a replication fork is relieved by enzymes called: a. DNA polymerases. b. DNA ligases. c. DNA helicases. d. topoisomerases.
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Which of the following is most likely to be found in the innermost region of the plasma membrane? a. a protein containing primarily non-polar amino acids b. a protein with a large number of amino acids which don't contain carboxyl groups c. any molecule with a phosphate group d. large molecules that are hydrophilic e. none of the above
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Which of the following statements concerning elongation of DNA at the replication fork is false? a. The Okazaki fragments are joined by the action of DNA ligase. b. The leading strand is synthesized continuously in the direction of replication fork movement. c. Both strands are synthesized continuously at the replication fork. d. The lagging strand is synthesized in Okazaki fragments backward from the overall direction of replication.
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Which of the following statements is true of all known DNA polymerases? a. They require a preformed primer, and they possess helicase activity. b. They synthesize DNA in the 5′ to 3′ direction, and they possess primase activity. c. They synthesize DNA in the 5′ to 3′ direction, and they require a preformed primer hydrogen-bonded to the template. d. They synthesize DNA in the 3′ to 5′ direction, and they possess exonuclease activity.
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amino acid letter cose
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c, b, d, e, a, c, a, c, a, c, c, d, d, 2. The eukaryotic small ribosomal subunit initially binds at what structure on the mRNA? a. A Shine-Dalgarno sequence b. The poly A tail c. The 7-methylguanosine cap d. A TATA sequence e. An AUG initiation codon$c
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protein through nuclear pore complex
1)importin binds to NLS (nuclear localization signal) in the protein being "cargo-ed" to the nucleus 2) The interaction of this complex with Ran/GTP on the nuclear side of the pore causes the release of the nuclear protein (the target protein) 3) Ran/ GTP/ importin recycled (out to cytosol)
What is the rate of error frequency for simple hydrogen bonding of complementary base pairs during replication? How much does it increase with the selection of a correct base by DNA polymerase, proofreading by DNA polymerase, and further repair mechanisms?
1/100 - 1/1000 1/10^5 - 1/10^6 1/10^8 - 1/10^9
Nucleic acid phosphate group is attached at? Carries what charge>
5' Negative
Discuss the organization of the transcriptional regulatory regions on genes with reference to:
9 elements 1. upstream enhancer 2. promoter 3. promoter 4. tata 5. inr 6. intron 7. intron 8. AATAA 9. Enhancer
Describe the basic characteristics of the average human gene
9 exons 8 introns 5' untranslated region coding sequence 3'untranslated region very large intron
Introduction: three main categories of cytoskeletal proteins
: Actin filaments/ microfilaments vs. intermediate filaments vs. microtubules
What are the three adjacent tRNA binding sites in the ribosome called & what are their roles in translation?
A (aminoacyl) site: "Arrival" P (peptidyl) site: "Polypeptide" (Pause) E (exit) site : "Exit"
Describe the differences between ATP dependent vs. symport/ antiport mechanisms
ATP - uses atp for energy to pump smporter uses coupled transport in the same direction antiporter - coupled transport in opposite direction
Describe the three types of structures involved in cell-cell interactions:
Adhesion junctions, desmosomes, tight junctions, gap junstions cadherins - integral cell proteins adherin - actin filament desmosomes
- DNA looping influence transcription
Allows transcription factors to bind to distant enhancer and interact with proteins on the polymerase/mediator complex
Structure DNA and RNA
Base, sugare and phosphate group
Briefly describe how the Golgi apparatus is involved in the export of proteins and lipids from the ER; and how a particular protein (or "cargo") is selected for a particular vesicle
Both free and ER lumen proteins and membrane lipids travel the secretory pathway in vesicles - ER -ERGIC (intermediate compartment) - Golgi - out
-Define the term "cell"
Cells are the fundamental unit of life. life = • movement • metabolism • reproduction • response to environment
channel protein
Chanel proteins form open pores through membranes allowing molecules of appropriate size and stricture to pass
co-translational Step 5:
Complete protein (minus the SRP, which is not part of its 3D structure) enters ER lumen
, constitutive heterochromatin
Contains DNA that is not transcribed in any cell type
, facultative heterochromatin
Contains DNA that is not transcribed in the current cell but may be transcribed in other cell typrs
Type of nucleic acids
DNA , RNA, mRNA, tRNA, rRNA
Review the "central dogma" of cell biology the role of RNA and gene regulation in this process
DNA Replication-entire genome Transcription-fraction of genome expressed (included introns and exons) Translation-even smaller fraction of genome expressed (only exons of final spliced mRNA is involved)
isolation of transcriptional regulatory elements in the laboratory
DNA affinity chromatography used to isolate transcription factors based on binding to specific DNA sequeces agarose beads and high salt buffer
chromatin
DNA complex with protein packs DNA into small space
Briefly describe how DNA polymerases catalyze the synthesis of DNA during DNA replication in the 5' à 3' direction
DNA polymerase are a groups of enzymes. They add dNTP to a preformed primer strand h bonded to the template They add new dNTPs to the 3' end of a new DNA strand
Spacer sequence
DNA sequences between genes on a chromosome
What is the primary factor that determines the pathway in which a particular protein will be translated?
Destination
General function of nucleic acid
Genetic information storage, protein synthesys
• Triacylglycerol structure
Glycerol head OCH2-OCH-CH2O • Fatty acid tail
Briefly describe the snare hypothesis of vesicle fusion: actually, don't.
Just realize that the process of fusing one lipid-bilayer bound membrane to another is complicated and involves more than a simple "meshing" of the two bilayers. Don't memorize the last two slides of this objective. -Docking -Tethering -Fusion
How is LDL and its receptor differentially processed during endocytosis?
LDL and receptors taken from plasma membrane in clathrin coated pit -redeptor is taken back to plasma membrane - LDL travels in endosome to lysosome - cholesterol is released
NLS
Nuclear localization signal specific amino acid sequences that are recognized by transport receptors and direct the transport of proteins from the cytoplasm through the nuclear pore complex into the nucleus.
List the 4 basic macromolecules which comprise cells
Nucleic acid- possible RNA was first Proteins - Lipids - basic component of membranes Carbohydrates
- Carbohydrate functions
Nutrient or Storage
The bond between 5' phosphate and 3' hydroxyl group
Phosphodiester
- List the major organelles found in eukaryotic cells and their general functions
Plama membrane - selective barrier - Nucleus - contains genetic material - Mitochondria - oxidative metabolism - Chloroplast - plants, photosynthesis - Lysosomes - digestion of macromolecules - Peroxisomes - various oxidative reactions - Vacuoles - digestion, storage - Endoplasmic reticulum - process and transport of proteins, synthesis of lipids - Golgi apparatus - sort and transport proteins for secretion, lipid synthesis
• Fatty acid structure
Polar head with non polar tail
What is the significance (function) of the PABP binding to the 3' poly AA tail?
Poly A binding protein identifies the strand as a complete mRNA and holds 3' out of the way of binding with 40S subunit complex
Why is the reverse transcriptase activity of telomerase necessary to replicate the ends of chromosomes?
Polymerase cannot replicate the extreme 5' end of linear DNA
• describe the four levels of protein structure
Primary - sequence of AA • Secondary - localized folding (alpha helix/beta sheet) • tertiary - non localized folding interactions • Quaternary - interaction with other polypeptide chains
Define "fluid mosaic model", integral membrane protein
Proteins are afloat in a sea of lipids proteins can be trans membrane or anchored to the membrane with a fatty acid or glycolipid
Define the term "proteomics" and discuss why these "____ - omics" techniques represent a major advance in cell biology.
Proteomics is the large scale analysis of cell proteins. permits analysis at a subcellular level
why have red blood cells been used in the study of the plasma membrane?
RBC contain no internal membranes including nuclear membrane
What are the primary difference between the smooth and rough ER
RER - covered with ribosomes on outer surface and is involved in protein metabolism SER - no ribosomes. involved in lipid metabolism
central dogma of molecular biology,
Replication - transcription - translation DNA, mRNA, rRNA, tRNA, semi-conservative replication, genetic code. RNA molecules are synthesized from DNA templates, and proteins are synthesized from RNA templates.
ORI yeast vs. higher eukaryotes
Replication origin in yeast is a 100bd ARS ori in eukaryotes is less well defined and may involve aspects of chromatin structure
Exons
Segment of protein coding sequence
Carrier protein
Selectively bind and transport specific molecules ie glucose
SRP
Signal Recognition protein (small RNA particle) recognize and bind to signal sequences (i.e., specific amino acid sequences) in the growing protein while the proteins is still being translated (the "address" of where the protein will go to is coded in the protein itself). composed of small cytoplasmic non-coding RNA and proteins
What is an SRP/ SRP receptor? What are their functions?
Signal Recognition protein - binds to signal on emerging protein and takes complex to SRP receptor on ER.
co-translational Step 4:
Signal peptidase enzyme on inner ER membrane/ translocon recognizes the SRP and cleaves it off the growing polypeptide chain
Co-translational Step 1:
Signal sequence on N terminus of translating protein binds SRP
Monosaccharides
Simple sugars
Outline the basic steps involved in the initiation of translation in eukaryotes
Step 1: 40S ribosomal subunit + eIFs (euk. Initiation Factors) + charged tRNA (met) assemble Step 2: assembly from Step 1 meets up with a mature mRNA (that has a whole bunch of other eIF factors bound to it, don't memorize them except note the PABP) and binds to its 5'm7G cap. Step 3: Step 1 assembly + Step 2 assembly starts at 5'm7G cap and scans mRNA for AUG start codon Step 4: Entire complex dissociates from start site (except the tRNAmet ), 60S ribosomal subunit attaches to 40S + tRNAmet , translation starts.
Outline the basic steps involved in the termination of translation in eukaryotes
Step 1: stop signal (codon) eventually ends up in the A site. Step 2: a releasing factor (a protein called "Release/ Releasing Factor) "moves in to the A site. Step 3: poly-peptide chain released Step 4: ribosome disassembles from mRNA (subunits likely recycled)
• - general function (contrast to other cytoskeletal proteins): What is the primary role of intermediate filaments? How is their function different from actin and microtubules? How are antibodies to intermediate filaments used in the lab?
Structural role • variety of proteins • strongest • not involved in movement
Leading strand
Synthesises in a continuous manner 3'-5'
TATA box, TBP, etc.
TATA - DNA sequenc found in the promoters TAT binding protein - basal transcription factor that binds to the TAT box
1. Point #3: Discuss (briefly) the basic order of transcription factor binding and RNA polymerase binding during the formation of the pre-initiation complex
TATA binding proteins TBPs - TFIID,TFIIF 2. RNA Pol 3. TBPs TFIIE, TFIIH
Key regulatory elements : Basal or core promoter (TATA Box, Inr)
TATA box and initiator (Inr) DNA sequence
TAF
TBP Associated Factors
Discuss how the structure of the plasma membrane of a cell is dictated by the amphipathic properties of phospholipids
The hydrophilic heads face the aqueous areas while the hydrophobic tales face the inside of the membrane
define transcription
The making of a mRNA copy of a gene by RNA Pol II
transcription factors
Transcription factors can function as either transcriptional activators and transcriptional repressors Transcription factors, like other proteins, have a modular structure-distinct structural domains carry out distinct functions Transcription factors contain separate DNA-binding and activation/repression domains. Transcription factors also contain other domains that regulate their activity or allow them to interact with other transcription factors and with the preinitiation complex
Remembering translation vs transcription
Transcription- RNA synthesis using a DNA template Translation-protein synthesis using an mRNA template dictionary definition: Transcription-process of making a copy (ie a document, text, ect) Translation-to turn from one language into another
the basics of translation
Translation is carried out on ribosomes, with transfer RNAs (tRNAs) serving as adaptors between the mRNA template and the amino acids being incorporated into protein (allows for change in "language"). mRNA read 5' to 3' the first amino acid is methionine (AUG)
. Of the following statements concerning microtubules, which one is FALSE? a. The location at which new microtubule formation originates in any one cell is based solely on where in that cell the microtubules are required. b. Dynamic instability of microtubules is determined in part by the rate of GTP hydrolysis. c. Polarity of microtubules is important for determining the direction of movement of dynein-based transport mechanisms. d. Microtubules play an important role in movement of vesicles within a cell.
a
10. Of the following statements concerning mRNA splicing in eukaryotic cells, which one is TRUE? a. Splicing is a two step process that requires different snRNAs to assist in carrying out the splicing reactions. b. Pre-mRNAs go through an intermediate stage in the splicing process when the poly(A) tail interacts with the 7-methylguanosine cap. c. The splicesome removes exons and ligates introns together to form the final mRNA. d. snRNPs are complexes consisting exclusively of protein, and make up the splicesome. e. mRNA splicing occurs predominantly in the nucleus, except for the those mRNAs encoding proteins required in the cytoplasm.
a
6. Short segments of newly synthesized DNA on the lagging strand are called a. Okazaki fragments. b. replicons. c. origins of replication. d. lagging fragments.
a
The use of reporter genes (GFP) to track expression of other proteins
a gene that is visualized when translated is placed upstream of the gene you actually want to visualize. tattle tale/tracker Reporter gene is a gene that encodes an easily assayed enzyme or other protein, and can be used to replace the normal gene sequence adjacent to a gene promoter through recombinant DNA technologies in a laboratory.
What different functions do cytoskeleton elements have?
actin - cell movement, dynamic structure intermediate - procide strength microtubules - railroad tracks in cell, cell movement
What is similar/ different about the structures of cytoskeleton elements?
actin - smallest diameter made of actin intermediate - made of preoteins microtubule - largest, hollow core
1. list the 3 main tenets of the "Cell Theory".
all organism composed of one or more cells 2. Cell is the structural and functional unit of life 3. cells can arise only by division from preexisting cell
3. Which of the following properties of water make it the ideal solvent for cellular activities? a. Water has a low heat of vaporization. b. Water is a polar molecule that can form hydrogen bonds with itself and with other polar molecules. c. Water dissolves nonpolar molecules. d. Water expands when it freezes.d e. none of the above
b
What processes can ultimately regulate the level of a protein in the cell? (see slides on "regulation of protein levels in the cell" and "protein degradation".
controlled at the level of transcription and translation determines the amount of enzyme (protein) synthesized by cell and how quickly degraded
1. What are the two major steps that must correctly occur in order for proteins to be targeted to their correct cellular location?
correct protein into the correct vesicle 2. vesicle to its correct destination
13. The important functional domain(s) of the matrix adhesion protein fibronectin is (are) a: a. collagen-binding domain. b. cell binding domain. c. proteoglycan binding domain. d. all of the above e. a and c only
d
14. Of the following statements concerning transcription factors, which one is FALSE? a. gene-specific transcriptional activator proteins can function by modulating the activity of the basal transcription complex (preinitiation complex) b. gene-specific transcriptional activator proteins can function by recruitment of HAT c. gene-specific transcriptional activator proteins can function by assisting in the basal transcription complex (preinitiation complex) assembly or disassembly gene-specific transcriptional activator proteins can activate gene expression independently of the general transcription factors
d
4. The genomes of saalamanders contain ten times more DNA than the genomes of humans. What would most likely account for this? a. Salamanders genomes have ten times as many protein coding genes as humans. b. Genomes increased in size during earlier evolution of all invertebrates and non-mammalian vertebrates, but then reduced in size during mammalian evolution. c. Salamander chromosomes contain unique types of histones that require unique DNA sequences found only in salamanders. d. Salamander genomes have a greater amount of non-coding and repetitive DNA sequences.
d
1. Both prokaryotic and eukaryotic cells a. carry out translation of proteins on ribosomes using DNA templates b. carry out transcription of proteins on ribosomes using RNA templates c. are separated from their external environment by a carbohydrate bilayer membrane d. use DNA as genetic material and RNA as a carrier of information e. use protein as genetic material and RNA as a carrier of information
d,
)/ What happens to lysosomal function in Tay-Sachs disease?
deficiency of beta-N-hexoamidase A which prevents degredation of ganglioside leads to buildup ganglioside and impaired function and death
Why is the term "secretory pathway" misleading?
does not just secret proteins from cell but sorts and carries both free and membrane bound proteins to intracellular destinations
Passive transport
does not require energy
colchicine and clcemid
drugs that block microtubule growth and mitosis
5. Why is yeast (Saccharomyces cerevisiae) an ideal model for cell biology? a. a. it has a genome with a similar number of genes as the human genome b. b. it can be readily grown in the laboratory, and has a rapid cell division time c. c. many of the fundamental mechanisms that regulate cell growth, division, and function in yeast are the same as in multicellular eukaryotic organisms d. d. all of the above b and c only
e
Primase,
enzyme that synthesizes short fragments of RNA as primer - complementary to lagging strand -5'->3' synthesis
What type of role does the extracellular matrix for epithelial vs. connective tissue? What are the general differences in composition between epithelial vs. connective tissue extracellular matrix? Bone vs. cartilage?
epithelial - thin sheetlike extracellular matrix called basal lamina. holds skin to body Muscle adipose nerve tissue - surrounded by thin basal lamina Connective tissue - ECM is prinicipally responsible for from and function of tissue. Collogen major structural protein
What other differences are there between eukaryotic vs. prokaryotic translation? How do the initiation signals differ?
euk require a greater numbet of none ribosomal proteins for initiation prok bind/start at shine-delgrano sequence euk 40S subunit finds 5'tg cap and slides along intil it gets to AUG start codon
Objectives #2- #4: Describe the basic structure and functions of the three major classes of macromolecules located in the extracellular matrix:
extra cellular matrix have common organization Tough fibrous structural proteins embedded in gel like polysachatides (groud substance) Adhesion proteins anchor cells in ECM
nuclear lamina
fibrous mess that provides structure proteins called lamins
post-translational modifications (review)
fold, multisubunit assembly, cleaved, phosphorylation, dephosphorylation, glycosylation, lipid addition. taged with ubiquitin
• Describe the basic characteristics (in terms of charge/ polarity, abilities to form bonds, etc.) of proteins
formed of amino acids (20) • AA can have side chains - nonpolar,polar, + charge, - charge • AA joined by peptide bonds • series of AA called polypeptides
• general vs. gene specific
general - involved in all polymerase II promoters. involved in formation of the transcription initiation complex • Gen specific - many different. bind to promoters and enhancers. direct activity of general transcription factors
basal or core promoter (TATA Box)
general TFs must bind to the Basal promoters in order to start transcription
Why is the final destination of a given protein to a specific subcellular compartment generally associated with the pathway it came from?
generally cytosolic pathway produces prteins for the nucleus, mitochondria, chloroplasts, peroxisomes secretory pathway - plasma membrane, secretory vesicles, endosomes, lysosomes, nuclear membrane
Describe the relationship of organism complexity to genome size and chromosome number
generally increased complexity = increased genome size but not direct relationship the smaller the genome the more of it is used for protein coding
protein degraded by
half lives (minutes/days) faulty proteins recognized and degraded
What is ground substance? How are the cells attached to their ECM?
in gel like polysachatides (groud substance) Adhesion proteins anchor cells in ECM
chromatin remodeling factors
increase availability of gene by displacing the nucleosome
What are the concentration and electrochemical gradients of Na+, K+, and Cl- with respect to the inside vs. outside of the cell?
large K inside - negative charge large NA and CL outside - positive charge
spliceosomes
large complexes composed of RNAs and proteins. Formed with several snRNA
nuclear pore complex
large macromolecular complex that differs significantly from typical membrane channels and transporters
proteasomes
large multi-subunit protease complexes that recognize and degrade polyubiquinated proteins
ion pumps
maintiain gradients across the plasma membrane,
aggrecan
major proteoglycan in cartilage
ubiquitin
marker in euk cells that targets proteins for degredation (proteolysis)
. What are lysosomes?
membrane enclosed organells that contain an array of enzymes for breaking down polymers at acidic pH acidity maintained by proton pumps
What amino acid is always translated first?
methionine (AUG)
centrosome
microtubule organizing centre. neg end of microtubule initiate microtubule growth
histone acetylation
modification of histones by the addition of acetyl groups to specific lysine residues in histone tails. results in transcriptionally permissive chromatin, whereas histone deacetylation gives rise to a non-permissive state
transcriptional co-activators and co-repressors work
modify the structure of chromatin to make its state permissive or non permissive for transcription
- role in intracellular transport: What is kinesin/ dynein? What do these molecules do?
motor proteins Minesins - towards bothe ends dyeins - to neg end only
why is facilitated transport not "active" transport?
moves with gradient. no energy expended
- How are intermediate filaments involved in disease?
mutant mice with missing keratin. Skin blisters similar to epidermolysis bullosa simplex in humans
Describe what is meant by DNA synthesis occurring in the 5' à 3' direction
new dNTP are added to the 3' end of the new strand by binding the dNTP 5' side to it
Introns
non-protein coding sequence
What are the steps in the elongation stage of eukaryotic translation?
o Step 1: tRNAmet IS PLACED IN the AUG start site (sits in P site) by the ribosome as the ribosome/ tRNAmet complex slides down the mRNA from the 5'm7G cap to the start site of translation (the AUG codon) o Step 2: the second (next tRNA) comes to A site o Step 3: a peptide bond is formed between the first two amino acids o Step 4: the ribosome moves downstream (so all the next tRNAs move from A site to P site after each peptide bond is formed o Step 5: repeat steps 2-4
Where does all (as far as we know, all) protein synthesis start?
on free ribosomes. Some remain free. Some finish the synthesis bond to the ER two branches of sorting (cytosolic vs secretory
What are the differences between pinocytosis, phagocytosis, and (receptor mediated) endocytosis?
pinocytosis - cell drinking phagocytosis - cell eating receptors are taking in witht eh coated vesicle but recycle through seperation in an early endosome
- TBP associated factors (TAF)
polypeptides associated with TBP in the general transcription factor - TFIID, TFIIB, TFIIF, TFIIE, TFIIH
Briefly discuss some of the most common types of post-translational modifications of proteins including:
polypeptides must form into distinct three dimensional shapes and assembled with other chains folding cleaving covalent modification phosphorylation glycosylation lipid addition
Describe the structure of tRNA and how tRNA serves as an adaptor between the "genetic code" (3-base triplet on an mRNA) and the correct amino acid
possess unique 3 nucleotide identifying sequences (anticodons) that allow the correct amino acid to be aligned with the appropriate codon in mRNA. tRNAs are approximately 70 to 80 nucleotides long and have characteristic cloverleaf structures based on intramolecular base-pairing
- Describe the environmental requirements thought to be necessary for formation of the first cell
presence of organic molecules (carbon based - molecules for catalysis of chemical reaction and sef replication - A barrirer between the internal cell and the external environment
Cell culture: primary cultures, immortal cell cultures
primary cell culture - first cultures established from tissue (limited number of divisions) Immortal cell lines - embryonic stem cells or cells from tumors that divide indefinitely in culture.
What is fibronectin, and in what sort of tissue would you find it?
principle adhesion protein of connective tissue
What is laminin, and in what sort of tissue would you find it?
principle connective tissue of the basal lamina
What is elastin? What sort of structures are you most likely to find it in?
principle protein in elastic fibres cross linked into a network by covalent bonds
Define the term "gene expression"
production of a particular mRNA from a gene
What does polycistronic vs. monocistronic mean? How is it relevant in eukaryotes vs. prokaryotes?
prok mRNA may be able ot translate for multiple proteins (polycistronic) Euk mRNA can translate for only one protein (monocistronic)
Key regulatory elements : Cis-acting DNA sequence
promoter and enhancer elements "Dimmer switch" for the gene to regulate transcription A promoter is the DNA sequence with which RNA polymerase and other proteins interact to initiate and regulate the rate of transcription of a gene.
of post-translational modifications of proteins including:
protein folding, cleavage, multiple polypeptide chains/ subunit assembly, covalent modifications -
kinetochore
protein structure associated with the centromere . microtubules bind act as molecular motor in mitosis and meiosis
What are ribosomes? Where are they located?
protein synthesis machines. in the cytosol and attached to membrane bound organelles
Briefly describe the difference between these two pathways
proteins are synthesized by free ribosomes in the cytoplasm. secretory - start synthesis on free ribosomes. finish synthesis in membrane bound organelles
Transcription factors and their activity:
proteins that are required for RNA polymerase II to initiate transcription
What is the signal hypothesis?
proteins that are synthesized on free ribosomes are moved to their final desitnation through recognition of targeting amino acid sequences within the protein
What are tight junctions? Do they provide strong or weak structural support?
provide seal between epithelial cells
Nuclear pore complex
provides the selective traffic of proteins and RNA through the nuclear envelope - 8 fold symmetry - 30-50 nucleoporins - parts cytoplasmic filaments / ring spoke ring nuclear ring / basket
DNA and RNA are composed of
purine and pyrimidine bases linked to phosphorylated sugars
Key regulatory elements : Upstream promoter/ Enhancer
regulatory sequences that can be a distance from the promoter
How is the speed of replication increased?
repeated rounds of DNa replication that produce multiple copies of a region
ced 3 and ced 4
required for apoptosis
Free vs. membrane-bound ribosomes:
ribosomes are located in the cytosol. some are free and some are located on membrane bound organelles
What are the similarities and differences between prokaryotic vs. eukaryotic ribosomes?
same general structure euk more complex proc 50 and 30 S subunits euk 60 and 40 S subunits
• nuclear envelope
separates the nucleus from the cytoplasm • two pl bilayers • perinuclear space between • nuclear lamina (protein framework • nuclear pore complexes Selective traffic of proteins and RNA through nuclear pore complexes
telomeres
sequeces at the end of eukaryotic chromosomes maintain stability of chromosomes telomere sequences are similar with cluster of G residues Shortened after each division Loss of repeats lead to unstable chromosome
Key regulatory elements : DNA sequence element
short sequence of DNA base pairs in a gene promoter with specific property with respect to regulation of transcription is ascertained
Okazaki fragment
small pieces of new lagging strand DNA that are joined to form intact DNA
Why is it important that the lumen of the ER is topographically equivalent to the exterior of the cell?
so that the domains of plasma membrane proteins that are exposed on the cell surface correspond to the regions of polypeptide chains that are translocated into the ER lumen
centromere
specialized region of the chromosome that ensures correct distribution of duplicated chromosomes during mitosis region siste chromatids are joined different size and sequences Have centromeric histone 3
sidetrack: describe how the smooth ER makes phospholipids for membranes (what is the role of flippase and why is it needed?
sue to hydrophobic nature new membrane lipids are synthesized in smooth ER Added only to the cytoplasmic facing leaflet Flipped to other side by flipase
Describe glucose transport as a two-step process which uses two transport mechanisms to get itself from the gut to the bloodstream
symporter with Na from intestinal epithelial cells Na pumped NA/K pump back out Glucose transported with facilitated transport through basolateral domain to blood
How does a tRNA align with the proper nucleotide triplet sequence?
tRNA has a unique 3 nucleotide identifying sequence that is aligned witht eh appropriate codon in mRNA
aminoacyl tRNA
tRNA with a specific amino acid covalently bound at the 3' end
mRNA
template for protein synthesis
In which direction is the DNA template read during DNA replication?
template is read 3' to 5'
peptide bond
the bond formed between the amino group of on AA and the carboxyl group of another AA dehydration (signle bond O and 2 H form H2O
What is a codon? What is an anticodon? What is an aminoacyl tRNA?
the codon is the triplet on the mRNA base pairs anti codon is on the tRNA and corresponds to specific condons aminoacyl tRNA is a tRNA with a specific amino acid covalently bound at 3' end
What is the "resting membrane potential" and why is it important for the activity of ion channels?
the ion gradient of a cell at rest
Describe the general structure and function of actin filaments (microfilaments) including: their assembly and the process of polymerization and depolymerization
the major cytoskeleton protein of cells
Describe the difference between how a protein that will be released into the lumen of the ER vs. being inserted into the ER membrane occurs.
the protein has a stop transfer sequence that bidns to the side of the translocon. Cahnges the conformation of the translocon to create a channel releasing the polypeptide chain to the ER membrane
Replication fork
the region of DNA where the parental strands separate and daughter strands are formed Active replication site containing the macromolecular complex
What is the wobble hypothesis?
there are more codons than there are amino acids. This allows for different codons to cod efor the same amino acid
What are coated vesicles?
those coated with cytosolic coat proteins
connexin
transmembran proteins that form a gap junction. 6 form a connexon
molecular chaperones: explain what a molecular chaperone is and its function, and why they are important (what might happen to a polypeptide chain during translation if there are no molecular chaperones?
very few proteins can properly self assemble within the cellular environment. chaperons are proteins that facilitate folding and translocation of other proteins. No information is provided
Where and how are ribosomes assembled?
within the nucleolus - pre-ribosomal particles from nucleus and proteins from cytoplasm. (1) imported to the nucleolus and then (2) attach to the pre-rRNA; (3) the pre-rRNA splits in two; one part becomes the 40S; the other becomes the 60S Sent back to cytoplasm as 40s and 60 s ribosomes REMEMBER: ribosomes = Protein + rRNA Both contribute to tertiary structure of ribosome
- steroid hormone receptors and homedomain proteins are examples of transcription factors
zinc finger domain regulate gene transcription in response to hormones - contain helix-turn-helix DNA binding play role in gene expression during embryonic development
