EC6: tRNA, aminoacyl-tRNA synthases, and ribosomes

Question 1

What is the role of tRNAs?

Answer 1

They deliver amino acids to the ribosomes for polymerisation into proteins

Question 2

What does ‘cognate’ mean?

Answer 2

Corresponding

Question 3

What structures do the 5’ and 3’ ends of the tRNA molecule form?

Answer 3

A helicial stem (‘panhandle’) with a tetranucleotide swinging arm at the 3’ end

Question 4

Why is the tertiary structure of tRNA (as revealed by X-ray diffraction) L-shaped?

Answer 4

Due to co-axial stacking of the D stem onto the anticodon stem and the T stem onto the acceptor stem

Question 5

How does tRNA interact with mRNA?

Answer 5

Triplet pairing on antiparallel strands

Question 6

What are aaRS enzymes and what do they do?

Answer 6

They are aminoacyl-tRNA synthases

They add amino acids to the correct (cognate) tRNAs

Question 7

What is crucial to ensuring the fidelity of gene expression?

Answer 7

aaRS

Question 8

Describe the experiment that emphasised the point that aaRS enzymes are crucial to ensure the fidelity of gene expression.

Answer 8

An amino acid attached to the correct (‘cognate’) tRNA was chemically modified to change it to one of the other proteinogenic amino acids (cysteine was reduced to alanine).

This incorrectly loaded tRNA was then introduced into a cell-free protein-synthesising system.

The Ala was incorporated in place of Cys in the polypeptide chain, showing that tRNA carries what it’s given and doesn’t recognise the amino acid it’s carrying

Question 9

Describe the two steps of aminoacyl-tRNA synthase action. By what are they driven?

Answer 9

1. Activiation of the amino acid
   aa + ATP + E ⇌ [aa-AMP-E] + PPi
   (PPi → 2Pi using pyrophosphatase)
2. Charging of the tRNA
   [aa-AMP-E] + tRNA ⇌ aa-tRNA + AMP
   This process is similar to charging a battery – adding something to something else

Driven by the hydrolysis of PPi by pyrophosphatase

Question 10

What do ribosomes consist of?

Answer 10

RNA plus protein

Question 11

How are ribosomes often referred to?

Answer 11

By their sedimentation coefficients (‘S values’)

Question 12

What are the two types of ribosome (bacterial and eukaryotic)?

Answer 12

70S (bacterial) and 80S (eukaryotic)

Question 13

What are the 70S and 80S ribosomal subunits?

Answer 13

70S = 50S + 30S

80S = 60S + 40S

Question 14

Why do the S values of the ribosomal subunits not add up to the S values of the ribosomes?

Answer 14

There is no reason why they should – the shapes of the subunits affect their sedimentation rate

Question 15

What are the proportions of proteins and RNA in all ribosomes?

Answer 15

One-third protein, two-thirds RNA

Question 16

Do ribosomes have active sites?

Answer 16

Yes – they are enzymes

Question 17

What happens on the ‘decoding site’ of the smaller ribosomal subunit?

Answer 17

Successive mRNA codons are recognised by incoming aminoacyl-tRNAs

Question 18

Where on a ribosome does peptide bond formation occur?

Answer 18

On the peptidyl transferase centre on the larger ribosomal subunit

Question 19

Where is the nascent polypeptide conducted?

Answer 19

Away from the subunit interface down the exit tunnel, which passes through the body of the larger subunit

Question 20

Describe the process of protein synthesis with reference to the ribosome.

Answer 20

As the ribosome migrates 5’→3’ along the mRNA during translation, successive mRNA codons are presented into the decoding site, located within the A site on the smaller ribosomal subunit

The ribosome maintains the fidelity of the gene expression by proofreading the choice of incoming aaRS into the vacant A site

Aminoacyl-tRNA is only accepted into the A site if its anticodon can form a cognate pair with the resident mRNA codon, otherwise the tRNA is rejected

Question 21

Which protein factors are also involved in translation?

Answer 21

• Initiation factors (IFs) – speed up the start of ribosomal protein synthesis
• Elongation factors (EFs) – travel with ribosome the whole way along the mRNA and are involved in each cycle
• Release factors (RFs) – used in association with ribosome to get rid of peptide chains

Ribosomes still work in the absence of protein factors, but not very well.

Question 22

How is energy required for mRNA translation generated?

Answer 22

Via GTP hydrolysis