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Bio 209 > Lecture 5B - Translation > Flashcards

Lecture 5B - Translation Flashcards

1
Q

How important is translation from an energy standpoint?

A

Cells devote more energy to protein synthesis than any other aspect of metabolism

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2
Q

How do four DNA bases encode the sequence of proteins that contain 20 different amino acids?

A

Using triplet code (codon)

4^2 = 16 AA, but 4^3 = 64 AA

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3
Q

What is evidence for triplet code?

A

Method: in vitro translation system (isolated translational components)

  1. Synthetic trinucleotides are sufficient to stimulate binding of aminoacyl-tRNAs to ribosomes
  2. Synthetic dinucleotide repeating mRNAs direct synthesis of polypeptides w/ 2 different AA

3, Synthetic trinucleotide repeating mRNAs direct synthesis of three different polypeptides w/ each containing the same AA

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4
Q

Triplicate codon sequences must be read in the _______ ________ ________, otherwise frame shift occurs => nonfunctional errors

A

Correct reading frame

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5
Q

7 properties of the genetic code

A
  1. Nucleotide is triplet based (Codon)
  2. Code is degenerate or redundant
  3. Code is non-overlapping
  4. Code is comma-free
  5. Code is ordered
  6. Code contains start and stop codons
  7. Code is nearly universal
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6
Q

What does it mean to say code is degenerate/redundant? What does this mean for tRNA?

A

Several codons encode the same AA; all but 2 AA are encoded by more than 1 codon

tRNAs can bp w/ more than 1 codon or there is more than one tRNA for many AA (both true)

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7
Q

What does it mean to say code is non-overlapping?

A

Nucleotides belong to only one codon

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8
Q

What does it mean to say code is comma-free?

A

Codons are read consecutively

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9
Q

What does it mean to say code is ordered?

A

Codons for same AA are similar, and codons for chemically related AA are similar

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10
Q

What does it mean to say codon contains start and stop codons?

A

AUG: start codon

UAG, UAA, UGA: stop codons

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11
Q

What does it mean to say that code is nearly universal?

A

Codons have the same meaning in all organisms

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12
Q

What is Crick’s wobble hypothesis?

A
  • Base-pairing rules only apply for the first two nucleotides of the codon
  • Base-pairing of the third nucleotide of codon is less stringent => this site can wobble
  • Inosine on tRNA can base pair w/ uracil, cytosine, or adenine
  • Allows for degenerate, but ordered genetic code
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13
Q

Describe AA activation/tRNA “charging”; Where does the AA bind? How many aminoacyl tRNA synthetases?

A
  1. AA is adenylated
  2. Aminoacyl tRNA synthetase catalyzes reaction b/t adenylated AA and tRNA to make AMP and aminoacyl-tRNA, matching the correct AA for the correct tRNA

AA binds at carboxyl carbon to 3’ OH of tRNA ribose

There is 1 aminoacyl tRNA synthetase (30-40) for each AA

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14
Q

What is hydrolytic editing of tRNA synthetase?

A

A trial by error mechanism - if AA is incorrect, it fits into the editing site and is cleaved from tRNA

Similar to DNA pol

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15
Q

Know the sizes (in S units) of euk and prok intact ribosomes and ribosomal subunits (small & large); What does the S mean?; Why is total different from subunit S?; which subunit binds to mRNA first?

A

Prokaryotic ribosome sizes: 30S + 50S = 70S

Eukaryotic ribosome sizes: 40s + 60S = 80S

S is Svedberg unit

Total is different due to sedimentation during centrifugation)

Small subunit binds mRNA first

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16
Q

Sites for tRNA binding to ribosome; how does ribosome move along mRNA

A

A-site: binds incoming aminoacyl tRNA

P-site: site of incorporation of new AA to growing polypeptide chain; binding site for peptidyl-tRNA

E-site: binding site for tRNA release after growing polypeptide chain has been transferred to aminoacyl-tRNA

Ribosome moves via translocation

17
Q

What catalyzes peptide bond formation in ribosome (proks vs euks)? Which subunit moves first?

A

Peptidyl transferase, a part of large ribosomal subunit (23S in proks, 28S in euks)

Large subunit moves first, followed by small subunit
(refer to slide 28, lec 5B)

18
Q

Ribosome composition by mass relative to rRNA and proteins?

A

Ribosome is 50/50 rRNA and ribosomal proteins

19
Q

Ribosomes have how many proteins and rRNAs?

A

> 50 proteins and 4 rRNAs

20
Q

Where are ribosomes located in proks vs euks? What happens to ribosome subunits after translation?

A

Proks: located throughout cell

Euks: located in cytoplasm, frequently on rough ER

After translation, subunits dissociate

21
Q

What are tRNAs in the context of translation machinery? How many versions?

A

Adaptor molecules that deliver amino acids to ribosome where it H-bonds to mRNA template

40-60 different versions

22
Q

Ribosomes undergo _____ and ______ with each round of protein synthesis

A

Association; dissociation

23
Q

What 4 things comprise the complex translation machinery?

A
  1. Ribosomes
  2. tRNAs
  3. AA activating enzymes (aminoacyl tRNA synthetase)
  4. Numerous other proteins involved in: initiation, elongation, termination
24
Q

Eukaryotic vs Prokaryotic translation initiation

A
  1. Initiator AA is methionine but not formylated
    - Proks have formylated methioinne
  2. Initiator complex forms at 5’ end of mRNA (recognizes 7-MG cap and reads until it hits AUG start) vs prokaryotic Shine-Dalgarno start site
25
Q

What is the significance of a formylated methionine initiator AA in prokaryotes?

A

The N terminus is capped via attachment of a 1C formate group => prevent unintended addition of polypeptide to that initiator AA

26
Q

Eukaryotic initiation details

A
  • The 7-MG cap on 5’ end of mRNA bound by cap-binding protein
  • Other initiation factors bind CBP followed by 40S subunit
  • Methionyl-tRNA (initiator AA) interacts w/ soluble initiation factors and binds ribosomal P site
  • Complex moves 5’ to 3’ on mRNA searching for start codon
  • Once at start codon => initiation factors fall off and 60S subunit binds Met-tRNA:mRNA:40S complex
27
Q

Purpose of Cap-binding protein

A

Helps ribosome determine if mRNA is undamaged and allows small subunit scanning of AUG start to being

28
Q

3 Steps of Chain Elongation

A

Same in proks and euks

  1. Aminoacyl-tRNA binds to A site
    - Elongation factor Tu (EF-TU) + GTP
  2. Peptide bond formation
    - Peptidyl transferase activity of 23S rRNA in proks, 28S rRNA in euks + GTP
  3. Translocation of ribosome to next codon
    - EF-G + GTP
29
Q

Function of EF-Tu

A

Escorts aminoacyl-tRNA to A site

  • Charged tRNA bound to EF-Tu interacts w/ A site
  • When correct codon-anticodon interaction occurs, EF-Tu binds to factor-binding center

-EF-Tu hydrolyzes bound GTP and is released

30
Q

Function of EF-G, what is ribosomal translocation

A

Stimulates ribosome translocation

Translocation: for each round of peptide bond formation
- P-site tRNA moves to E site
- A site tRNA moves to P site
- mRNA moves by 3 nucleotides to expose next codon
- EF-G hydrolyzes GTP => facilitates translocation

31
Q

Translational Termination

A
  • Sequence specific (UAA, UAG, or UGA)
  • When stop codons enter A site => Release factors enter and alter peptidyl transferase activity => H2O added to COOH terminus => releases peptide chain
32
Q

Initiation factors, elongation factors, and release factors

A

IFs: bind CBP following 40S (small) subunit, interact w/ Met-tRNA to bind P-site, fall off once at start codon

EFs: EF-Tu and EF-G

RFs: enter A site with stop codons, altering peptidyl transferase activity and adding water to COOH terminus => release peptide chain

Bio 209 (10 decks)

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