Lecture 15

Question 1

What are the requirements for translation?

Answer 1

1. mRNA
2. ribosomes
3. Charged tRNA
4. initiation factors
5. GTP

Question 2

Basic components of translation?

Answer 2

1. activation of the monomer
2. initiation
3. elongation
4. termination
5. processing the polymer

Question 3

How does tRNA become charged tRNA?

Answer 3

1. enzyme bound amino acid- adenylate
2. Formation of the aminoacyl-tRNA

reaction is driven by the hydrolysis of pyrophosphate

Question 4

What is the first AA in prokaryotes and in mitochondria?

Answer 4

N-formylmethionine (fMet)

Question 5

What is the role of fMET?

Answer 5

It allows for initiation

Question 6

Prokaryotes have two tRNA’s for methionine?

Answer 6

1. one allows for the formation of fMAT

2. allows for AUG codon

Question 7

Two tRNA’s that recognize AUG in eukaryotes?

Answer 7

1. The first codon also uses MET, and it has a special tRNA for this first codon (but the MET amino acid is not
   formylated) .
2. Normal MET for internal codons

Question 8

What is the purpose of the Shine Dalgarno sequence?

Answer 8

This sequence is purine rich

found in prokaryotes to allow for correct alignment of the AUG codon

Question 9

How do eukaryotes align the mRNA with the ribosome?

Answer 9

lack Shine Dalgarno sequence

eukaryotic small ribosome binds close to the cap at the 5’ end, scans until it encounters the AUG start codon.

Question 10

The steps to protein synthesis -initiation?

Answer 10

1. Initiation factors (IFs) aid in the formation of the 30S initiation complex.
2. The charged initiator tRNA is brought to the P site of the 30S subunit by IF-2-GTP.
3. GTP on IF-2 is hydrolyzed and initiation factors are released when the 50S subunit arrives to form the
   70S initiation complex

Question 11

Protein synthesis- elongation

Answer 11

1. Elongation factor EF-Tu-GTP brings the appropriately charged tRNA to the codon in the empty A site (decoding). GTP on EF-Tu is hydrolyzed.
2. Peptidyltransferase activity of the 23S rRNA of 50S subunit catalyzes peptide bond formation, transferring
   the initiating amino acid (or peptide chain) from the P site to the amino acid at the A site (transpeptidation).

Question 12

protein synthesis - translocation

Answer 12

1. EF-G-GTP facilitates movement of the ribosome three nucleotides along the mRNA in the 5’ to 3’
   direction.
2. Steps are repeated until a termination codon is encountered at the A site.

Question 13

Protein synthesis- termination

Answer 13

A termination codon is recognized by a release factor (RF-1 or RF-2), which results in release of the newly synthesized protein. GTP on RF-3 is hydrolyzed. The synthesizing complex dissociates.

Question 14

In prokaryotes the mRNA is what?

Answer 14

polycistronic

contains multiple genes

Question 15

What is the toxin Diphtheria toxin?

Answer 15

inactivation of EF-2 by ADP-ribosylation

this prevents translocation during protein synthesis

Question 16

Streptomycin

Answer 16

Prevents assembly of ribosome (binds to 30s subunit)

Question 17

Tetracycline

Answer 17

Block elongation by preventing aminoacyl-tRNA access to the A-site

Question 18

Erythromycin

Answer 18

Binds to the 50S subunit of the complete (70S) ribosome

prevents ribosome translocation

Question 19

Chloramphenicol

Answer 19

Inhibits peptidyl transferase activity in prokaryotes

At high levels, may inhibit mitochondrial translation

Question 20

Cycloheximide

Answer 20

Inhibits eukaryotic peptidyl transferase activity

Question 21

Puromycin

Answer 21

Causes premature termination of translation in both prokaryotes and eukaryotes

Question 22

What are the 6 major post-translational modifications?

Answer 22

1. protein folding
2. covalent alterations
3. proteolytic processing
4. addition of prosthetic groups
5. prenylation
6. protein degradation

Question 23

zymogen activation

Answer 23

Activation by enzymatic cleavage of peptide bonds of the zymogen molecule

nonactive to active

Question 24

protein phosphorylation by a kinase

Answer 24

phosphate is transferred from ATP to AA side chain

occurs on OH groups

most common post-translational modification

Question 25

What are the two types of glycosylation?

Answer 25

1. N-linked- addition of N to asparagine | 2. O-linked- sugar is attached to serine or theonine

Question 26

Lipid anchoring?

Answer 26

The cell targets Ras protein to the cytosolic face (inner leaflet) of the plasma membrane by a lipid anchor mechanism - with the aid of farnesyl groups.

Question 27

What protein is essential for proper insulin folding?

Answer 27

C-peptide

Question 28

Proteolytic Processing of Insulin

Answer 28

1. gene for insulin is transcribed into mRNA 2. translation of that mRNA 3. Translation of the polypeptide is directed into the lumen of the rER and forms “preproinsulin” 4. The signal sequence is cleaved in the lumen of the rER – forms “proinsulin” 5. Proinsulin is moved from the rER to the golgi, where it is cleaved to form insulin and C-peptide 6. insulin and C-peptide go into secretory vesicles 7. secreted by exocytosis