Lecture 14 - The ribosome

Question 1

What is the rate of translation?

Answer 1

10-20aa per second

Question 2

What are the A, P, and E sites on the peptidyl transferase centre?

Answer 2

The A site is where the incoming amino acylated tRNA can attach.

The P site harbors the polypeptide chain

The E site is the exit site of the deacylated tRNA.

These sites are common to both subunits.

Question 3

Explain the structure of the 30s subunit.

Answer 3

0.8 MDa in size
21 proteins on the surface
1500 nucleotides rRNA

Question 4

Explain the function of the 30s subunit.

Answer 4

Initiates binding to the mRNA via initiation factors at the Shine-Dalgarno sequence.

Ensures correct translation takes place via the decoding center

Question 5

Explain the structure of the 50s subunit.

Answer 5

1.5MDa in size
34 proteins on the surface
3000 nucleotides of rRNA

Question 6

Explain the function of the 50s subunit.

Answer 6

Peptide bond formation occurs here

Question 7

What domain in the 50s subunit houses the peptidyl transferase center?

Answer 7

Domain V (5)

Question 8

What are the properties of ribosomal proteins?

Answer 8

It is made up of 55 proteins.

Most proteins have one or more globular domains

Globular domains located on the surface, extensions and loops buried within the structure.

They recognize RNA through shape and charge complementarity.

Question 9

What are initiation, elongation and termination factors in translation?

Answer 9

Initiation factors: Assemble ribosome onto the mRNA

Elongation factors: Permit production of the polypeptide chain.

Termination factors: Halt protein synthesis

Question 10

Explain the ribosomal stalk model

Answer 10

bL12 (also known as L7/L12) form a complex with L10 and L11.

The H43/H44 helices form a platform upon which L10 and L11 bind.

Question 11

Name two elongation factors and their function.

Answer 11

Ef-Tu: Delivers aminoacyl tRNA to the ribosome

EF-G: Promotes translocation of tRNAs during synthesis

They bind to the bL12 stalk. (ribosomal stalk region)

Question 12

Where is the ribosomal exit tunnel located and what are its dimensions?

Answer 12

It is located within the 50s subunit

100A in length, 20A width.

The tunnel wall is lined with 23s rRNA and is constricted by loops.

It also permits limited folding.

Question 13

Explain how different regions in the exit tunnel are associated with different folding events.

Answer 13

The upper/central part of the tunnel is quite narrow, so it can support the formation of turns and basic alpha helices.

The lower part of the tunnel is wider, so it can form structures such as beta hairpins and alpha helices.

Any large 3D structure will form outside the tunnel.

Question 14

Explain the mechanism and biochemical evidence of the arrest motif SecM.

Answer 14

SecM is an arrest motif 17 amino acids long.

Amino acids within the SecM stall motif interact with the ribosomal RNA, which then relay back to the peptidyl transferase centre, preventing a tRNA from entering the ribosome, stalling translation elongation.

Biochemically, after treatment with RNaseA, a stalled ribosome will have an upshift of the band on the gel, as it is still attached to a tRNA.

Question 15

Explain the bifurcation in the ribosomal exit tunnel

Answer 15

Bifurcation occurs around uL23, at the lower half.

The nascent chain’s (NC) pathway is dictated by sequence and length.

Short NCs with unfolded segments are directed through pathway 2, with H6/H7 rRNA.

Long NCs with unfolded segments are directed through pathway 1, with H50/H24 rRNA.

Question 16

What is the structural basis of decoding?

Answer 16

When a near cognate codon binds, there is a flip seen in A1492 and A1493 residue of the helix 44.

When the correct (cognate) codon binds, a flip of G530 of the 530 loop on helix 18 is ALSO seen.

When the right codon binds, a ‘530 pseudoknot’ forms in the 16s RNA.

This 530 pseudoknot changes the conformation to interact with the codon-anticodon double helix’s minor groove and only interacts with the first 2 bases.

As a result, wobble base pairing seen in only 3rd base.

Question 17

Explain the codon sampling step.

Answer 17

The amino-acid tRNA complex is transferred to the PTC via EF-Tu*GTP

The anticodon on the tRNA samples the A-site to see whether there is a match with the mRNA codon

Question 18

Explain Codon recognition and GTPase activation

Answer 18

With a correct match, there is the 530 pseudoknot conformation change, and the tRNA shifts by 5 degrees.

A domain closure of the 30s subunit is triggered, causing changes in the loops of EF-Tu.

His84 in the loop exposed, and catalyzes GTP hydrolysis.

EF-Tu undergoes a conformational change, causing it to dissociate from the ribosome.

Question 19

Explain competing co-translational events.

Answer 19

Targeting by SRP at ~25 amino acid sequences

N-terminus processing by PDF and MAP at ~44 amino acid sequences.

Chaperone binding by Trigger Factor at ~100 amino acid sequences.

Question 20

Give an overview of trigger factor.

Answer 20

Trigger Factor (TF) is present in prokaryotes

It is 47 kDa in size, and binds to the ribosome at L23.

It has a 1:1 stoichiometry, and constantly cycles on and off the ribosome, with a mean residence time of 11-15s.

With the Nascent chain emerging, it has a mean residence time of 50s, and affinity for the ribosome increases 9-30x.

Question 21

Why would you want the ribosome to act as a holdase?

Answer 21

1. It hands the NC over to auxiliary proteins (eg. TF).
2. It protects the NC against misfolding.
3. It delays folding
4. It guides co-translational folding intermediates.

Question 22

What protein is used to study the ribosome as a holdase and why?

Answer 22

Alpha-synuclein is used, because it is an intrinsically disordered protein. As a result, interactions can be studied without any complications of folding .

It also contains a number of charged residues, so whether electrostatic interactions are involved in guiding interactions between the NC and ribosome.

Question 23

Explain the studies showing the interaction between NC and the ribosome.

Answer 23

Through 15N-spectra and molecular dynamics, we can see that most of the time, the NC is making contact with the ribosome via ribosomal proteins, however, it is also seen that the NC is able to make contact via 23s rRNA.

Contact is made approx. 45 amino acids from the exit tunnel.

Control: Alpha-synuclein + isolated 70s

Question 24

Explain the study showing the interaction between Trigger Factor and the NC.

Answer 24

Through 15N-spectra, it was seen that the NC region that interacts with the TF is the same as the region that interacts with the ribosome.

Control: Alpha-synuclein + isolated 70s + TF

Question 25

Explain the process of the ribosome as a holdase.

Answer 25

A cloud of Mg2+ ions allow transient interactions with the NC.

Negative NCs have increased local dynamics, positive and aromatic residues on NC cause reduced mobility.

As translation occurs positive and aromatic residues on NC can interact with the TF.