Cundliffe 5 translation protein processing

Question 1

INITIATION FACTORS

Answer 1

Prokaryotic = IF 1 IF 2 IF 3
Eukaryotic = many eIFs

Question 2

ELONGATION FACTORS

Answer 2

Prokaryotic: EF-Tu EF-G
Eukaryotic: eEF1 eEF2

Question 3

RELEASE FACTORS

Answer 3

Prokaryotic: RF1 RF2
Eukaryotic: single eRF

Question 4

INITIATION OF TRANSLATION IN BACTERIA

Answer 4

AUG not necessarily right at end => smallest subunit recognized (30s)

Question 5

Reading frame

Answer 5

= reading triplets from AUG, ribosome binds to mRNA

⇨ cover initation codon up to nucleotides & AUG

Question 6

Shine – Dalgarno reconition [S-D]

Answer 6

RNA interaction =complementary recognistion of codons & cause synthesis to start at right place on mRNA

Question 7

Functions of tRNA f

Answer 7

special initiator tRNA, cognate for methionine & AUG initiation codon
Only 1 Met R synthase = tRNA synthase puts Met on both tRNA.

Question 8

Functions of tRNA f

1

Answer 8

1 30s ribosomal subunits associate with 2 initiation factors & prevented from associating with 50s subunits, therefore free & available for initiation (for mRNA binding)

Question 9

Functions of tRNA f

2

Answer 9

2 Another IF Initiator aminoacyl-tRNA & GTP binds

Question 10

Functions of tRNA f

3

Answer 10

3 Products bind to mRNA => 30s initiation complex [30s subunit, mRNA, IFs, initiator aminoacyl-tRNA, GTP]

Question 11

Functions of tRNA f

4

Answer 11

4 50s ribosomal subunits join => triggers GTP hydrolysis, accompanies release of all 3 IFs => 70s initiation complex [ 70s ribosome, mRNA, initiator aminoacyl- tRNA]. Now complete with initiator aminoacyl –tRNA recognizing mRNA initiation codon on ribosomal P site.

Question 12

Protein initiation factor 2 IF2

Answer 12

tertiary complex happen away from ribosome.

Question 13

INITIATION OF TRANSLATION IN EUKARYOTES

Answer 13

Basic mechanics of ribosomal function similar

• NOT use [S-D] complex.
• many more Ifs,
• subject to de/activation via phosphorylation. => control point in eukaryogic gene expression.
• elongation & termination phases of translation as similar again.

Question 14

POLYPEPTIDE CHAIN ELONGATION

Answer 14

• initiator aminoacyl-tRNA inserted into P Site.
• binding of aminoacyl –tRNA enters A Site in response to mRNA codon downstream to last codon.
• Carried to ribosome as ternary complex w GTP & EF protein.
• GTP hydrolysed & EF released bound to GP.
• initiator A-tRNA now in Psite & A-tRNA in A site.

Question 15

Ribosome = 3 binding sites for tRNA

Answer 15

E, P, A

Question 16

Peptide bond formation:

Answer 16

transfer of nascent peptide: tRNA in Psite onto aminoacyl-tRNA in Asite.
- Elongated peptide attached to tRNA in Asite & deacylated (uncharged) tRNA remains I P site. - Amide bond Catalysed by ribosome. => to re-start cycle need translocation.

Question 17

Enzymes used in translation:

Answer 17

Aminoacyl-tRNA binding 
(EF Tu GTP)
Peptidy transferase
Translocation 
(EF G GTP)

Question 18

Translocation reaction

Answer 18

move through ribosome so that next downstream mRNA codon can get to A site for decoding in next cycle. Codons are triplet nucleotides, movement must span exactly 3 mRNA nucleotides to maintain reading frame
Deacylated tRNA moves Psite => E site & peptidyl-tRNA moves A site => Psite

Question 19

Translocation reaction Catalysed by

Answer 19

Growing peptide chain along exit tunnel. Catalysed by elongation factor & involves GTP hydrolysis.
Next aminoacyl-tRNA enters A site = new cycle
- A& E sites are mutually exclusive, whole chain is translated from P site => A site.

Question 20

POLYPEPTIDE CHAIN TERMINATION

Answer 20

3 stop codons: not normally recognized by tRNA => peptidyl-tRNA in P site, mRNA presents stop codon in A site

Question 21

Termination codon recognized by

Answer 21

RF (release factor) causes peptide to be released from ribosome.
Eukaryotes = 1 large RF, binds to GTP & catalyses CTP hydrolysis. Bacteria= 2 RF’s RF1 & RF2, also for GTP hydrolysis

Question 22

After polypeptide released

Answer 22

ribosome leaves mRNA & dissociates subunits to bind to next mRNA => change partner subunits for each reaction.