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Molecular Biology Flashcards

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1
Q

What are the four requirements of DNA-templated DNA?

A
  1. Single-stranded template
  2. Deoxyribonucleotides with 5’ triphosphate
  3. Magnesium ions (essential co-factor for the polymerase)
  4. Annealed Primer (often RNA) w/ a free 3’ OH
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2
Q

What is the DNA polymerase reaction mechanism?

A
  1. Incoming triphosphate nucleotides form W-C bonds w/ template strand
  2. Magnesium stabilizes 3’OH + alpha phosphate
  3. Bond forms btw alpha phosphate and 3’OH
  4. Beta and Gamma phosphate are released as PPi new phosphodiester bond is formed
  5. As long as the template strand is available - a new substrate is generated
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3
Q

How does shape discrimination contribute to base-pair geometry to the fidelity of DNA replication?

A
  • the active site has enough flexibility to enable catalysis of properly matched bases
  • incorrect base pairing - contortion of binding pocket to form H-bonds btw the template and the incoming nucleotides - less favourable
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4
Q

How is error correction by DNA polymerase I done?

A
  • movement is inhibited when an incorrect nucleotide is added
    intrinsic 3’-5’ exonuclease proofreading activity - permits the enzyme to remove a newly added nucleotide
  • mismatched base pair will favour the exonuclease domain over the extension domain
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5
Q

How does DNA accomplish with high-fidelity?

A
  • errors lead to mutations - not typically good
  • complementarity btw bases pairs favoured - leads to correctness
  • shape discrimination of active site
  • proof-reading activity - incorrect bases
  • repair pathways
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6
Q

What is the fractionation experiment?

A
  1. start w/ a cell or organism that contains your protein of interest
  2. break it open under conditions that preserve the proteins (cold, proteases)
  3. soluble or insoluble?
  4. separate it based on: size, solubility, hydrophobicity, charge
  5. Confirm activity via an activity assay
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7
Q

What are Kornberg’s experiments?

A

Assay to demonstrate whether/not the isolated fraction contains DNA polymerase
1. Mixed radio-labelled dT (method 1) or radio-labelled dTTP (method 2 w/ isolated fraction
2. add acid to the reaction + separate the reaction mixture into 2 fractions: a) acid soluble 2. acid insoluble by centrifugation
3. acid insoluble mixture could be polymerized DNA, including polymerized
4. Verified acid-insoluble pellet -> formed at the bottom of a centrifuge tube - acid-resistant
DNase is added - real nucleotide polymers would be broken down
thus supernatant would contain polymerized DNA

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8
Q

What is the main function of DNA polymerase I?

A
  • most abundant; insufficient for replication
  • primary function - clean up during replication, recombination and repair
  • distinct 3’-5’ proofreading exonuclease - performs nick translation by mild protease treatment separate - Klenow fragment
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9
Q

What is the main function of polymerase II, III, IV, and V?

A

DNA polymerase II: DNA repair
DNA polymerase III: principal replication enzyme in E. coli
- responsible for rapid and accurate duplication of the genome
DNA polymerase IV and V: translation -repair damage from UV radiation

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10
Q

What is Nick translation?

A

important in:
- DNA repair and the removal of RNA primers during replication
DNase - single stranded cuts (nicks)
Treated with DNA polymerase I which performs two reactions:
1) additions of new nucleotides in 5’ to 3’ directions
2) Removal of nucleotides in 5’ to 3’ direction
Gets labelled

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11
Q

What is Meselson-Stahl experiment?

A

Semi-conservative: Parent and daughter DNA
addition of nitrogen 15 into grown E.coli - makes DNA heavy, which is then collected after some replication it is added to nitrogen 14 media (Light) - allows cells to divide - newly synthesized
Separate the DNA by density
Heavy - parent strand
intermediate - acting as a template strand
intermediate and light -> semi-conservative
Heavy and light - conservative

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12
Q

How is DNA replicated bi-directionally?

A

Replication fork: dynamic points where parent DNA is being unwound and separated
Separation and replication are done simultaneously
Both ends have active replication forks
can be bi-directional or uni-direction

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13
Q

How is replication initiated?

A

Denaturation mapping: selective denaturing of sequences unusually rich in A=T pairs to provide landmarks along the DNA molecules - generates of single strand bubbles

origin: location where replication loops are initiated

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14
Q

What are the fundamental rules of DNA replication?

A
  • semi-conservative
  • begins at an origin and proceeds bi-directionally
  • synthesis proceeds in a 5’ to 3’ direction and semi-continuous
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15
Q

What is the experimental approach to distinguish which model of replication?

A

3 possibilities: semi-discontinuous, discontinuous, pieces
How can these models of replication be distinguished:
1) Method to detect newly synthesized DNA - Okazaki “pulsed” E.coli w/ radio-active thymidine - newly synthesized DNA would be hot
2) Determine the size of newly synthesized DNA - alkaline sucrose density gradient to separate DNA based on size
3) Monitor this reaction of a time scale suitable for observing intermediates in DNA synthesis - assay fraction @ various time points - once cells are lysed you halt DNA synthesis - “snap” shots

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16
Q

Why can’t DNA polymerase replicate the genome alone?

A
  1. separate the DNA strand - done by DNA helicases
  2. stabilize the single-stranded DNA - the energetic cost of exposing those N-rich bases
  3. start replication w/out a free 3’OH - primase add a short RNA primer to leading strand and in many stretches on the lagging strand - DNA-RNA hybrid provides something like a substrate for DNA polymerase
  4. too slow and not processive enough to replicate
  5. cannot replicate in 5’-3’ direction
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17
Q

What are the four requirements of DNA-templated DNA polymerase?

A
  • single-stranded template
  • deoxyribonucleotides w/ 5’ triphosphate (dNTPs)
  • Magnesium ions (essential c-factor for the polymerase)
  • annealed primer (often RNA) w/ free 3’OH
    synthesis only occurs in the 5’-> 3’ direction
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18
Q

How does the DNA polymerase III clamp loader function?

A

responsible for the faithful replication of the genome - one for the leading strand and lagging strand
clamp-loading complex and AAA+ ATPase
binding ATP and new β Sliding Clamp
clamp loaders are composed of Three τ Subunits Along with the δ and δʹ Subunits - which contribute to the overall function
conformational change opens the ring at one subunit interface
AAA+ ATPase domain hydrolyzes ATP to ADP which closes the ring

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19
Q

How is leading and Lagging strand synthesis?

A
  1. helicase - unwinds DNA at the replication fork
  2. The leading strand is synthesized in one piece
  3. The lagging strand is synthesized in fragments
  4. Primase adds a primer to generate a free 3’OH
  5. Clampe loader - ensure synthesized is processing
  6. processing until primer on older Okazaki fragment
  7. clamp loader - re-loaded on new primer/template RNA-DNA hybrid unit synthesis is complete
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20
Q

What are the 2 new problems after the synthesis of short fragments of DNA?

A
  1. gaps btw the end of the fragment and the new RNA primer
  2. short regions of RNA-DNA hybrid complexes in lagging strand
    DNA pol I - removes the RNA and DNA replaces it with/ DNA through the specialed exonuclease activity
    DNA ligase seals the remaining nick
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21
Q

What is the endogenous DNA damage?

A
  • Polymerase errors
  • spontaneous deamination reaction
  • spontaneous hydrolysis of base
  • oxidative damage
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22
Q

What is the exogenous DNA damage?

A
  • UV photo-damage
  • Alkylating agents
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23
Q

What is spontaneous deamination?

A

Deamination: spontaneous loss of exocyclic amino groups
- cytosine to uracil
- uracil chemically similar to thymidine
- left unrepaired -> A-T mutation
In DNA -> uracil is foreign and removed

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24
Q

What are depurination reactions?

A

depurination: hydrolysis of the N-
β-glycosyl bond btw the base and the pentose
- creates an apurinic or apyrimidinic site or abasic site
- commonly w/ purines

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25
Q

How is DNA damaged by oxidative damage?

A
  • reactive oxygen species (hydrogen peroxide, hydroxyl radicals, superoxide radicals) damage DNA
  • hydroxyl radicals -> most oxidative DNA damage
  • defence system against oxygen species
    Guanine -> 8-oxo-guanine
  • anti conformation is favoured - new steric clash between carbonyl and ribose oxygen now inc. hoogsteen base (syn conformation) pairing btw G and A
    after replicating -> A-T
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26
Q

What is UV radiation and how does it affect DNA?

A

Promotes pyrimidine dimers
- cyclobutane pyrimidine dimers
- 6-4 photoproduct
ionizing radiation causes:
- ring opens
- base fragmentation
breaks in the covalent backbone of nucleic acids

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27
Q

What is alkylation damage?

A

O6-methylguanine: modified nucleotides that form in the presence of alkylating agents
- common and highly mutagenic lesion
- pairs w/ T rather than cytosine

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28
Q

What are the mutations linked to cancer?

A

mutation: a permanent change in the nucleotide sequence
substitution mutation: replacement of one base pair w/ another
insertion mutation: the addition of 1+ base pairs
deletion mutation: the deletion of 1+ base pairs
silent mutations: affects nonessential DNA/negligible effect on gene function.

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29
Q

What is base-excision repair (BER)?

A

DNA glycosylases: recognize common DNA lesions and remove the affected base by cleaving the N-glycosyl bond
- abasic site -> removal of a base by DNA glycosylase
uracil DNA glycosylases -> removes uracil from DNA which resulted from spontaneous deamination of cytosine
other DNA glycosylases:
- formamidopyrimdine
- 8-hydroxylamine
- hypoxanthine
- pyrimidine dimers

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30
Q

What is the mechanism of base excision repair?

A
  1. A damaged base is recognized by a specified glycosylase that removes the base
  2. endonuclease cleaves the phosphodiester backbone at the basic site
  3. DNA pol I replace missing base (short extension)
  4. Gap filled w/ DNA ligase
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31
Q

What is nucleotide-excision repair in E.coli + humans?

A

repairs DNA lesions that cause large distortions in the DNA helical structure
excinuclease: hydrolyzes two phosphodiester bonds, one on either side of the distortion
DNA pol I or DNA polymerase ε (humans) fills
DNA ligase seals the nick

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32
Q

What is the direct repair of alkylation damage?

A

O6-methylguanine-DNA
methyltransferase: catalyzes the transfer of the methyl group O6-methylguanine to a cysteine residue
- single methyl transfer - permanently methylates the protein (inactivating it)

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33
Q

What is MGMT?

A

this enzyme binds to alkylated guanine residues - flips the base out and removes the methyl group - restoring the guanine to its canonical W-C binding
suicide enzyme - not regenerated
expensive - preservation of genome code

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34
Q

How are Alkylated bases directly repaired?

A

AlkB transfers a hydroxyl group from alpha-ketoglutarate to the methyl group which can then be released as formaldehyde restoring the chemistry of the original base

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35
Q

What is non-homologous end joining?

A

Resection - degradation can take place on either side of the break - single-strand overhangs
Ku70/80 complex recruited to either end and further recruited DNA-PKcs (kinase) subunits
Then recruits Artemis (nuclease) which - gets phosphorylated by DNA-PKcs and trims the single-stranded overhangs
Ligase IV, XLF, XRCC4 -> fixation of the double-strand break
Error-prone - insertion and deletion of base pairs

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36
Q

What is homologous Recombination?

A

repair - break w/ high fidelity - dividing stage -> S1 and G2 phase - no impact to genetic code
resection - single-strand overhang
2. binding of RPA (replication protein A) - ssDNA overhang
3. displacement of RPA w/ RAD51
4. BRCA2-mediated repair filament
5. BRACA1/BARD1 mediated strand invasion

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37
Q

What are TALENs?

A

Fokl nuclease: engineered to be target to specific regions of the genome by attaching to engineered DNA-binding motifs that will recognize specific sequences of DNA
- labour intensive

38
Q

How does genome editing treat/prevent HIV?

A

HIV to enter T-cells it must bind to the CD4 receptor and CCR5 co-receptor
- The variant of the CCR5 that is missing 32 base pairs in an exofacial loop - HIV cannot these cells

39
Q

What is the mechanism of CRISPR Cas9 genome editing?

A
  1. disordered PAM recognition domain
  2. binding of the TracRNA and crRNA (sgRNA) induces folding of the PAM recognition region
  3. PAM is the target sequence
  4. CRISPR-cas9 complex -> melts target DNA at the PAM
  5. sgRNA (crRNA) -> complementary to the target DNA it will continue to unwind and expand the R-loop
  6. Fully complementary the HNH domain moves over the cute site on the 3’ strand
    RuvC domain over 5’ strand and hydrolyzes the backbone
    - reorganization of the HNH domain during the binding of sgRNA and target - cleavage only possible w/ the change
40
Q

What are the similarities between RNA polymerase and DNA polymerase?

A
  • only 5’->3’ direction
  • requires magnesium as a co-factor
  • hydrolysis of incoming nucleotide-releasing PPi powers the rxn
  • W-C base pairing btw the template and incoming nucleotides
41
Q

What are the differences between RNA polymerase and DNA polymerase?

A
  • does not need a primer
  • no 3’->5’ editing (more error-prone)
  • W-C base pairing btw the template + incoming nucleotide except U instead of T
  • NTPs (deoxy)
42
Q

What is important for RNA polymerase holoenzyme?

A

Holoenzyme (RNA pol and sigma) - bind to promoter
an σ 70 (most common subunit) - directs the enzyme to specific DNA binding sites
σ 70-> activates RNA polymerase
- drives both conservation promoter sequences and spacing btw them
- after a small stretch of polymerization - sigma leaves complex
- NusA replaces it - elongation occurs until the end

43
Q

What is the ratchet mechanism?

A

Elongation
- bridge helix stabilizes and orients in-coming nucleotide
- makes W-C bonds w/ template
- triggers the loop swings closed - catalysis of the phosphodiester bond
- confirmation change shifts bridge helix - induces a wedge structure of the trigger loop - push against newly added nucleotide
-translocation and returns to the initiation state

44
Q

What are the 3 RNA polymerases in eukaryotic transcription?

A

RNA pol I: Pre-ribosomal RNA
RNA pol II: mRNA, ncRNA
RNA pol III: tRNA, 5S rRNA , ncRNA

45
Q

What is the function of RNA pol II?

A

responsible for the synthesis of mRNA and many ncRNAs
promoters sequences:
- TATA box -> near -30
- an initiator at +1

46
Q

What is the importance of TFII protein?

A

required for initiation of transcription and elongation - several TFII proteins

47
Q

Mechanism of transcription

A

initiation - TFII proteins and TFID and RNA pol II to promotor region (closed
- bending of the promotor helps open DNA at these regions
- Release of initiation factors and phosphorylation of CTD (serine 5 and 7 phosphorylated throughout transcription) drives elongation
- TFIID binds the promotor DNA in an elongated complex which anchored by TBP-DNA interaction on one end
initiate sequence is straddled on both ends by TFIID

48
Q

What is the footprinting technique?

A

identifies the DNA sequences bound by a particular protein
- radio-labelled at one and treated w/ Dnas (-lane)
- Mixed w/ predicted DNA binding protein before DNase treatment - binds to region (+lane)

49
Q

What is 5’cap of mRNA?

A

uniquie 5’-5 linkage
RNA pol CAP-synthesizing complex associates with the CTD
- guanine is added to RNA -> at the phosphate end and it is methylated on N7, as are the next two bases at the 2’OH

50
Q

What is polyadenylation?

A

coupled to transcription termination
- occurs at the 3’end of the mRNA
- transcribed - coding region serves as a binding site for cleavage recognition factors and requires two consensus sites
- cleavage is downstream
- adenine is added to the 3’ end
- stabilizes the RNA molecules and adds to nuclear export

51
Q

Pre-mRNA

A

proteins complexes that carry out mRNA-processing reactions are organized in association w/ each other and with the phosphorylated CTD of pol II
splicing removes the introns and ligates the exons together

52
Q

What is the spicing mechanism of group II introns?

A

OH group (adenosine) makes a nucleophilic attack - 5’ splicing site form a lariat structure
- 3’ OH of the 5’ exon acts as a nucleophile, completing the reaction
- lariat structure (contains adenosine) has three phosphodiester bonds

53
Q

What is a spliceosome and small nuclear RNA (snRNAs)?

A

a large complex made up of multiple specialized RNP complexes small nuclear ribonucleoproteins (snRNPs) and dozens of other proteins
- snRNAs: U1, U2, U4, U5, U6 are abundant in the nuclei

54
Q

How are U1 and U2 snRNA binding?

A

U1 snRNA - base pairs w/ the 5’ splice sites
U2 snRNP - base pairs w/ the 3’ end and identifies the A residue that becomes the nucleophile (BP=branch point)

55
Q

spliceosome assembly

A
  • addition of the tri-snRNP (U4, U5, U6 snRNPs) forms the spliceosome
  • requires ATP
  • single turnover enzymes
    mediating splicing - after leading the U2/U5/U6 RNA complex onto the intron several structural reorganization events occur, which are largely quality control steps to ensure the proper site has been identified
56
Q
A
57
Q

What is alternative splicing?

A

process in which a particular exon may or may not be incorporated into the mature mRNA transcript
more than one site where poly(A) tails can form (poly(A) site)
inc. variety of proteins generated from the genomes

58
Q

Ribosomal RNAs and tRNA undergo processing

A

pre-ribosomal RNAs: longer precursors of ribosomal RNA Ribothymidine - important in tRNA structure

59
Q

Transfer RNAs

A

RNase D at 3’ end cute and P at 5’ end cut
Base modifications
- 5’ cleavage
- 3’ cleavages
-CCA addition
Splicing -> The bottom part of the intermediate is spliced - involved in H bonds with mRNA
modifications: methylation, deamination or reduction

60
Q

What are exosomes?

A

large 3->5 exoribonucleases in eukaryotes that are responsible for RNA degradation
- broken down into nucleotides and re-used

61
Q

What is a reading frame?

A

method of dividing nucleotides such that a new codon begins every 3 nucleotide residues
- set when the translation of an mRNA molecule begins
- maintained as triplets are read sequentially
missense protein:
- The initial reading frame is off by one or two bases
- translation skips a nucleotide in the mRNA

62
Q

What is the initiation codon?

A

initiation codon (AUG): signal the beginning of a polypeptide

63
Q

What are the termination codons?

A

Termination codon: (UAA, UAG, UGA) normally signals the end of the polypeptide synthesis
- do not code for any known amino acids

64
Q

What is a missense mutation?

A

mutation in which a single new base pair replaces another

65
Q

What is silent mutation?

A

mutation in which the nucleotide is different but the encoded amino acid stays the same

66
Q

what is a transition mutation?

A

involves the replacement of one purine base with another purine or one pyrimidine base with another pyrimidine

67
Q

What is the core structure?

A

phosphate backbone, pentose sugar and nitrogen-rich bases

68
Q

How do purines have a slight pucker?

A
  • beta-furanose = found in nucleotide and nucleoside forms of the bases
  • pucker can be found in 2-exo or 3-exo conformation
  • changes the orientation of the base and the 5’carbon - impacts the orientation of the phosphate
  • 2’OH group of ribose favours C’3 endo
  • 2’H group of deoxyribose favours the C-2’endo conformation
69
Q

What is the 1’N-glycosidic bond?

A
  • btw base and pentose at the 1’C
  • anti-conformation
70
Q

What is the Watson-Crick model of DNA?

A
  • polar sugar-phosphate backbone on the outside
  • nitrogen-rich hydrophobic bases inside
    C - G -> 2H bonds
    A - T -> 3H bonds
  • antiparaellels strands are complmentary
71
Q

How are polymers stabilized?

A
  • delocalized electron sharing btw the planar bases in the z-dimension -> base stacking
  • hydrophobicity of the nitroge-rich bases drives the formation of helical structures to occlude water from the interior -> hydrophobic effect
  • hydrogen bonds -> stabilize helix
  • metal ions -> shield the negative charge of the backbone
72
Q

What is the Hoogsteen “non-canonical” base-pairing?

A
  • rotation around the N-glycosidic bond - new face to form H-bond
  • facilitate H-bonds btw nucleotides from both sides of the base forming a branched structure
    Ex. T-A and Wobble (G-T) and Guanine (anti)-adenine(syn)
73
Q

How are mRNA structures complex?

A
  • single-stranded
  • forms a right-handed helix
  • dominated by base-stacking
    complementary region of DNA or RNA
  • paired strands are antiparallel
74
Q

How is tRNA structured?

A
  • canonical 3D fold enabling it to be charged w/ an amino acid so that it fits the ribosome during peptide synthesis
  • clover-leaf structure
    1. Atypical H-binds w/ 2’OH
    2. Hoogsteen interaction and W-C interaction
    3. Modified bases
75
Q

What are the modified bases?

A

5-Methylcytidine: common modification involved in gene regulation
Inosine: tRNA in codon recognition
Pseudouridine: uracil base w/ an alternative N-glycosidic linkage critical in RNA folding

76
Q

What is site-directed mutagenesis?

A
  • study the impact of a small change on the structure/function of proteins we need to make a change to
  • requires DNA polymerases and primers
77
Q

What are the biophysical properties?
Chromatin vs DNA

A

Chromatin
- neg charge of the phosphate-sugar backbone - stabilized by pos changes histone protein
- reduced steric repulsion + enables being + flexible polymer properties

Naked DNA
- Negative charge -> phosphate backbone
- requires metal ions

78
Q

What is the Sky-FISH experiment?

A
  • specific sequences of DNA w/in a chromosome are labelled w/ a fluorophores
  • DNA is denatured -> anneals
  • visualized using fluorescence light microscopy
  • distinct domains of chromatin fibres from individual chromosomes form individual chromosome territories
79
Q

What is progeria?

A
  • C->T mutation that generates a cryptic splice site
  • removal of the end of the 11th exon generates a protein product that is 50 amino acids shorter than wt Lamin A protein
  • irregularities in the shape of the nucleus, epigenetic modification -> reduce mobility of the Lamin A protein
  • rescued by a small oligo that targets the cryptic exon 11/12 boundary -> sends this RNA product for generation
80
Q

Characteristics of tRNA

A
  • 8+ residues have modified bases and sugars
  • guanylate (pG) residue at the 5’ end
  • trinucleotide sequence CCA(3’) end
  • cloverleaf shape
81
Q

What is the amino acid arm?

A

carries a specific amino acid esterified by its carboxyl group to the A residue at the 3’end of the tRNA

82
Q

What is the anticodon?

A

contains anit-codon
anti-parallel to the mRNA, 3->5 so 1 starts at 3’ and mRNA 3 starts a 3’ this is the wobble position
inosinate -> the anticodon tRNAs include the nucleotide inosinate (designated I) -> weak hydrogen bonds w/ A, U, and C

83
Q

What is the main function of aminoacyl-tRNA synthetases?

A

catalyzes the tRNA and a specific amino acid
- occurs in the cytosol
- activates the carboxyl group of each amino acid
- links each new amino acid and the information encoding it in the mRNA
-“charged” when attached to their amino acid
“second genetic code” interaction between aminoacyl-tRNA synthetase and tRNAs

84
Q

Explain the proofreading by aminoacyl-tRNA synthetase

A

amino acids differ in small characteristic
- editing site of incorrect aminoacyl-AMP it gets hydrolyzed but the tRNA does not become aminoacylated to the wrong amino acid

85
Q

What is the mechanism of aminoacyl-tRNA synthetase reaction?

A

Step 1: forms the enzyme-bound intermediate, aminoacyl adenylate
- carboxyl of amino acid attacks alpha phosphorus of ATP forming 5’-aminoacyl adenylate

step 2: transfer the aminoacyl group from enzyme-bound aminoacyl-AMP to its corresponding specific tRNA
- class II aminoacyl-tRNA synthetases -> directly transfers to the 3’OH
- class II aminoacyl-tRNA synthetase -> transfers first 2’OH and then transesterification moves ti 3’OH

86
Q

Ribosomes

A
  • complex supramolecular machines -> large and small subunits -> made up of rRNA and ribosomal proteins
  • complexity=> chloroplast and mitochondria < bacterial ribosomes < eukaryotic ribosomes
  • requires R proteins
87
Q

What are the three steps of initiation of tRNA?

A

initiation in bacteria requires:
- 30S
- mRNA
- fMet-tRNA
- three initiation factors: IF1, IF2, IF3
- GTP
- 50S
-Mg2+

Step1: 30S subunit binds IF1 and IF3 (prevents the 10S and 50S subunits -> prematurely combining) then the tRNA
- shine-Dalgarno sequence: region on mRNA that guides the initiating (5’)AUG to its correct position
two Methionine
- fMet-tRNA inserts N-formyl methionine at the amino-terminal end
- Met-tRNA inserts methionine in interior positions

Step 2: anticodon of fMet-tRNA -> correctly paired w/ mRNA initial codon at Psite

Step 3: GTP hydrolyzes the initial complex and allows the 70S to bind

88
Q

What are the ribosome’s three sites of binding in tRNAs?

A

aminoacyl site (A): incoming aminoacyl-tRNA
- IF1 binds and prevents tRNA binding at the A site
peptidyl (P) site: amino acids are added to the growing chain
exit (E) site: binds uncharges tRNAs

89
Q

What is the elongation mechanism of tRNA?

A

Step 1:
- incoming aminoacyl-tRNA binds a GTP-bound EF-Tu binds a GTP-bound EF-Tu complex
- aminoacyl-tRNA-EF-Tu-GTP complex binds to the A site of the 70S initiation complex
- GTP hydrolysis, EF-Tu-GDP complex leaves

Step 2: Peptide bond formation
- N-formylmethionyl group transferred to the amino group of the aminoacyl-tRNA in the A site to form a dipeptidyl-tRNA

Step 3: Translocation
- EF-G- GTP => hydrolyzes and moves the site

90
Q

How is termination done?

A

termination signal codon -> UAA, UAG, UGA -> in A site
termination factors (release factors): RF1, RF2, RF3
functions:
- hydrolyzes the terminal peptidyl-tRNA bind
- release the polypeptide and last uncharge tRNA
- dissociation 70S

BIOC 302 Molecular Biology (1 decks)

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