Roper - Ribosomes

Question 1

How big is a ribosome?

Answer 1

It is a similar size to viruses

It constitutes 25% of the mass of a bacterium.

Question 2

What is a ribosome made of?

Answer 2

A ribozyme decorated with proteins.

Proteins modulate the properties of hte ribosome but RNA carry out the condensation reactions

Question 3

What does S stand for when describing the size of ribosomal subunits?

Answer 3

Svedberg, the inventor of analytical ultracentrifugation - it measures the buoyant molecular weight of biological molecules

Question 4

What subunits are prokaryotic ribosomes made up of?

Answer 4

30S and 50S to make 70S

Question 5

What subunits are eukaryotic ribosomes made up of?

Answer 5

40S and 60S to make 80S

these are larger and more complex that prokaryotes

Question 6

What are the three types of RNA molecules used by ribosomes?

Answer 6

tRNAs (transfer), rRNAs (ribosomal) and mRNAs (messenger)

Question 7

What drug targets ribosomes?

Answer 7

Puromycin

Question 8

What structural features do ribosomal subunits have that contributes to its function?

Answer 8

The 50S subunit has an A, P and E site.

The 30S subunit has the tunnel through which mRNA feeds.

Question 9

What is the A site?

Answer 9

where aminoacyl-tRNA bind, carrying the next amino acid

Question 10

What is the P site?

Answer 10

where peptidyl-tRNA (with phosphodiester bond already formed with the previous amino acid) binds

Question 11

What is the E site?

Answer 11

where the tRNA that was previously in the P site exits, now without any amino acid

Question 12

What is the anticodon stem loop?

Answer 12

where the tRNA interacts with mRNA

Question 13

How far apart is the anticodon stem loop from the site where peptide bond formation happens?

Answer 13

80 angstroms apart - very far which indicates how big the ribosome is and shows how the two processes are separated by huge molecular distances

Question 14

What does the anticodon stem loop always end in and why?

Answer 14

CCA

The phosphate on the 2 or 3’ hydroxyl of adenine (last base on tRNA molecules) is what binds to amino acids.

Question 15

What makes tRNA molecules so unstable?

Answer 15

tRNA synthetase puts amino acids on the 2’ phosphate on adenine but it can hop between both because they are chemically equivalent.

Question 16

Crystal ribosome structures were published in 2000. What contributed to the increase in resolution of ribosome structures before the 2000s?

Answer 16

• X-ray sources and computing.
• Crystal Quality and source.
• X-ray Detectors
• Crystal Freezing techniques
• Phasing strategies

Question 17

How has Electron Microscopy contributed to the increase in resolution of ribosome structures?

Answer 17

EM provided early micrographs, early ideas of shape.

Now Cryo-EM is the prominent technique to solve new ribosome structures

Question 18

What did we learn from early structures of the ribosome?

Answer 18

That the interface between the two ribosomal subunits is almost entirely RNA.
Proteins in ribosomes are unusual; they have a globular head domain which tends to be on the outside, with long meandering structures which reach into the core of the ribosome but none get within 20 angstroms from the peptidyl transferase site.

Question 19

What do we know about the structure of RNA in ribosomes?

Answer 19

Adenine residues are highly conserved in the interface between subunits and RNA helices.

Question 20

What is the RNA morphology in the 30s subunit?

Answer 20

the 16s rRNA molecule has a distinct morphological shape in the subunit where the 5’ end forms the body, the central part forms the platform, the major 3’end forms the head and the minor 3’ end straddles the interface.

Question 21

What is the RNA morphology in the 50s subunit?

Answer 21

the 23s rRNA is more intricately woven through the proteins to form a monolithic (strong, sturdy, singular) structure.

Question 22

What have the structures we learned about peptide bond synthesis from got in common?

Answer 22

came from bacterial ribosome crystallised at pH 6.0, more acidic than optimum pH of 7.5
this could affect the bases so structures need to be taken with pinch of salt

Question 23

What did Steitz and Moore 2003 show?

Answer 23

• Only RNA was needed - protein free 23s RNA could do it invitro later.
• Tunnel through the 50S where polypeptides go through as synthesised + end of the tunnel is where tRNA’s CCA end binds to mRNA.
• Photoaffinity labelling studies show that rRNA’s U-2584 and U-2585 are close to site of peptide bond formation - part of a highly conserved internal loop in the 5th domain of the 23sRNA

Question 24

What did Steitz and Moore 2003 show?

Answer 24

• Only RNA was needed - protein free 23s RNA could do it invitro later - closest protein is 18 Angstroms away
• Tunnel through the 50S where polypeptides go through as synthesised + end of the tunnel is where tRNA’s CCA end binds to mRNA.

Question 25

Who found structures for peptide bond formation?

Answer 25

Steitz and Moore 2003

Question 26

What did structures show about the site of peptidyl bond formation?

Answer 26

Two related inhibitors were used to locate the site of peptidyl transferase activity
Photoaffinity labelling studies show that rRNA’s U-2584 and U-2585 are close to site of peptide bond formation - part of a highly conserved internal loop in the 5th domain of the 23sRNA

Question 27

What is the mechanism?

Answer 27

The N3 nitrogen of Adenine 2486 takes a proton from the existing peptide chain. Normally its pKA is too low to lose it spontaneously. Then it can then undergo nucleophilic attack that forms the peptide bond.