L14: Eukaryotic protein synthesis

Question 1

Protein synthesis in prokaryotes

Answer 1

1. Activation of AA: the tRNA is aminoacylated
2. Initiation: mRNA & the aminoacylated tRNA bind to small ribosomal subunit. The large subunit then binds as well
3. Elongation: successive cycles of aminoacyl-tRNA binding & peptide bond formation occur until ribosome reaches a stop codon
4. Termination: translation stops when stop codon is encountered. mRNA and protein dissociate & the ribosomal subunits are recycled
5. Protein folding

Question 2

Polysomes (or polyribosomes)

Answer 2

Cluster of ribosomes, bound to mRNA molecule

Question 3

Molecular components for activation

Answer 3

Aminoacyl-tRNA synthetases: each cell carries at least 20 specific enzymes, each specific for AA. Each enzyme binds specific AA and matching tRNA

tRNA: specific binding sites on tRNA that are recognised by aminoacyl-tRNA synthetases

AA

ATP

Question 4

Aminoacyl-tRNA

Answer 4

Linkage to 3’ end of tRNA

Linkage to carboxyl AA

Question 5

Molecular recognition of codons in mRNA by tRNA

Answer 5

At opp end of tRNA: codon recognition site

Codon recognition site is a sequence of 3 bases: anticodon

Triplet of bases align itself in complementary fashion to codon triplet on mRNA

Question 6

Molecular components for peptide chain initiation in prokaryotes

Answer 6

mRNA

f-Met-tRNAifMet

30S and 50S ribosomal subunits

Set of proteins known as initiation factors

GTP

Question 7

mRNA

Answer 7

mRNA sequences that serve as signals for initiation of protein synthesis

Question 8

First codon is AUG (Met)

Answer 8

1. All organisms have 2 tRNAs for Met. In bacteria, plus chloroplasts and mitochondria initiation, tRNA inserts N-formylmethionine (uses tRNAfMet)
2. Eukaryote protein synthesis. Begins with Met, but still a tRNA is used

Question 9

Ribosome sites for tRNA

Answer 9

A, or acceptor, site

P, or peptidyl, site

E, or exit, site

Question 10

Initiation factors

Answer 10

IF-1: prevents premature binding of tRNAs to A site

IF-2: facilitates binding of fMet-tRNAfmet to 30S ribosomal subunit

IF-3: binds to 30S subunit; prevents premature association of 50S subunit; enhances specificity of P site for fMet-tRNAfMet

Question 11

Formation of initiation complex in prokaryotes

Answer 11

1. 30S subunit bunds IF-1 and IF-3, then mRNA
2. IF-2—GTP binds 30S subunit and recruits fMet-tRNAfMet, which bp with start codon
3. 50S subunit associates, IF-2 hydrolyses GTP and IF-1, IF-2, and IF-3 dissociate, leaving initiation complex

Question 12

Molecular components for elongation

Answer 12

30S and 50S ribosomes

Aminoacyl-tRNAs

Elongation factors

mRNA

GTP

Question 13

Molecular components for initiation

Answer 13

f-Met-tRNAifMet

mRNA

30S and 50S ribosomal subunits

Initiation factors

GTP

Question 14

Elongation

Answer 14

Aminoacyl tRNA binds to elongation factor Tu that also carries GTP

Aminoacyl-EF-Tu-GTP complex binds to aminoacyl (A) site for 70S initiation complex

After GTP hydrolysis, EF-Tu-GDP leaves ribosome

Peptide bond forms

Protein synthesis is N-terminal to C-terminal

Translocation: ribosome moves 1 codon toward 3’end of mRNA

Question 15

Molecular components for termination

Answer 15

Release factors

GTP

Question 16

Termination

Answer 16

Signalled by stop codon: UAA, UAG, UGA

Stop codon in A site -> trigger action of termination factors (release factors): RF-1, RF-2, RF-3

Question 17

Release factors

Answer 17

Hydrolyse terminal peptide-tRNA bond

Release peptide & tRNA from ribosome

Cause subunits of ribosome to dissociate so that initiation can begin again

Question 18

Ribosomal subunits

Answer 18

Cycle rapidly through protein synthesis

80% of ribosomes are engaged in protein synthesis at any instant

When 70S ribosome dissociates from mRNA -> separates into free 30S and 50S subunits

Intact 70S ribosomes are inactive in protein synthesis (only 30S subunits can interact with initiation factors)