Lecture 5B - Translation

Question 1

How important is translation from an energy standpoint?

Answer 1

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

Question 2

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

Answer 2

Using triplet code (codon)

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

Question 3

What is evidence for triplet code?

Answer 3

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

Question 4

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

Answer 4

Correct reading frame

Question 5

7 properties of the genetic code

Answer 5

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

Question 6

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

Answer 6

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)

Question 7

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

Answer 7

Nucleotides belong to only one codon

Question 8

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

Answer 8

Codons are read consecutively

Question 9

What does it mean to say code is ordered?

Answer 9

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

Question 10

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

Answer 10

AUG: start codon

UAG, UAA, UGA: stop codons

Question 11

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

Answer 11

Codons have the same meaning in all organisms

Question 12

What is Crick’s wobble hypothesis?

Answer 12

• 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

Question 13

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

Answer 13

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

Question 14

What is hydrolytic editing of tRNA synthetase?

Answer 14

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

Similar to DNA pol

Question 15

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?

Answer 15

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

Question 16

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

Answer 16

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

Question 17

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

Answer 17

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)

Question 18

Ribosome composition by mass relative to rRNA and proteins?

Answer 18

Ribosome is 50/50 rRNA and ribosomal proteins

Question 19

Ribosomes have how many proteins and rRNAs?

Answer 19

> 50 proteins and 4 rRNAs

Question 20

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

Answer 20

Proks: located throughout cell

Euks: located in cytoplasm, frequently on rough ER

After translation, subunits dissociate

Question 21

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

Answer 21

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

40-60 different versions

Question 22

Ribosomes undergo _____ and ______ with each round of protein synthesis

Answer 22

Association; dissociation

Question 23

What 4 things comprise the complex translation machinery?

Answer 23

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

Question 24

Eukaryotic vs Prokaryotic translation initiation

Answer 24

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

Question 25

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

Answer 25

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

Question 26

Eukaryotic initiation details

Answer 26

- 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

Question 27

Purpose of Cap-binding protein

Answer 27

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

Question 28

3 Steps of Chain Elongation

Answer 28

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

Question 29

Function of EF-Tu

Answer 29

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

Question 30

Function of EF-G, what is ribosomal translocation

Answer 30

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

Question 31

Translational Termination

Answer 31

- 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

Question 32

Initiation factors, elongation factors, and release factors

Answer 32

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