The Ribosome and Translation - Lecture 16

Question 1

What are the ribosomal units in prokaryotes?

Answer 1

○ Ribosome = 70S
○ Large subunit = 50S
○ Small subunit = 30S

Question 2

What are the ribosomal units in eukaryotes?

Answer 2

○ Ribosome = 80S
○ Large subunit = 60S
○ Small subunit = 40S

Question 3

Why do the subunits not add up?

Answer 3

○ S = a measure of the sedimentation rate of suspended particles centrifuged under standard conditions - shape and size
○ rRNA’s have elaborate secondary structures made up of 40 or more stem loop

Question 4

What are the three stages of protein synthesis?

Answer 4

○ Initiation
○ Elongation
○ Termination

Question 5

What does protein synthesis require?

Answer 5

○ Ribosomes
○ tRNA
○ mRNA
○ Accessory factors
○ Energy (ATP and GTP)

Question 6

How is an amino acid activated?

Answer 6

○ Amino acid is attached to 3’ tRNA acceptor arm
○ Amino acid used ATP to make an amino acyl-AMP
○ Amino aycl-AMP joints to tRNA to make amino acyl tRNA and AMP is ejected

Question 7

How is the right start codon found?

Answer 7

○ Prokaryotes: Shine-Dalgarno sequence
○ Eukaryotes: Kozak sequence
○ Interacts with 16s rRNA of the 30s small subunit to identify the site of protein synthesis

Question 8

Why are there two tRNA’s for methionine?

Answer 8

○ One for initation
○ One for elongation
○ Prokaryotic initiation tRNA is modified to be formylated

Question 9

How is translation initiated?

Answer 9

○ Initiator tRNA and mRNA join ribosomal complex using initiation factor IF2
○ Initiation factors/GTP bind to 30S subunit
○ Large subunit joins to complex with the P pocket situated over the first codon
○ IF3 dissociates
○ Inactive IF2 is ejected and GTP is hydrolysed to GDP

Question 10

What are the roles of the initiation factors?

Answer 10

IF1:
○ Blocks A site to initiator tRNA - met
○ Inhibits premature 30S - 50S interaction
IF2:
○ Tags initiator tRNA
○ Regulates entry into ribosome
IF3:
○ Inhibits premature 30S - 50S interaction
○ Stabilises free 30S
○ Accuracy check for initiator tRNA - met binding

Question 11

What does the elongation stage involve?

Answer 11

○ Ternary complex is brought to the A pocket by elongation factor
○ Ternary complex = aminoacyl tRNA + EFTu-GTP
○ EFTu gets ejected and GTP is hydrolysed to GDP
○ Peptide bond is formed
○ tRNA is ejected from P site and association of EG-G-GTP to ribosome
○ Ribosome translocates
○ EFG is ejected and GTP is hydrolysed to GDP

Question 12

What are the roles of the elongation factors?

Answer 12

○EF-Tu: mediates aminoacyl-tRNA entry to ribosome
○ EF-G: mediates translocation

Question 13

What does the termination stage involve?

Answer 13

○ RF-GTP binds to A site when termination codon appears
○ Hydrolysis of polypeptide chain from tRNA
○ Dissociation of tRNA and RF (release factors)

Question 14

What are the three release factors in prokaryotes?

Answer 14

○ RF1
○ RF2
○ RF3 - Helps RF1 or 2 bind to ribosome - GTPase

Question 15

What is the role of GTP in each stage of protein synthesis?

Answer 15

○ Initiation: addition of large subunit
○ Elongation:
- Addition of aminoacyl-tRNA by EF-Tu and EFG
- Peptide synthesis and translocation
○ Termination:
- Release of peptide chain
- Dissociation of ribosome