27 - The Ribosome

Question 1

What type of structure is a ribosome?

Answer 1

ribonucleoprotein particle (RNP)

More contemporarily: ribozyme

Question 2

What proportion of the ribosome is RNA? Protein?

Answer 2

• 2/3 RNA

- 1/3 Protein

Question 3

25% of the dried cell mass of an E.coli cells is?

Answer 3

Ribosomes, there are about 15,000 in a cell

Question 4

What are the rRNAs that make a prokaryotic ribosome? (3)

Answer 4

• 23S
• 16S
• 5S

Question 5

What are the proteins that make a prokaryotic ribosome? (3)

Answer 5

• L1
• L2
• L3

Question 6

What are the subunits that make a prokaryotic ribosome? (2)

Answer 6

• Large: 50S (5S+23S)

- Small: 30S (16S)

Question 7

What is the sedimentation coefficient of a prokaryotic and eukaryotic ribosome?

Answer 7

Prokaryotic: 70S
Eukaryotic: 80S

Question 8

All ribosomes contain two ________ (16S-18S, 23S-28S) and one or two small _____ (5S, 5.8S)

Answer 8

All ribosomes contain two major rRNA molecules (16S-18S, 23S-28S) and one or two small rRNAs (5S, 5.8S)

Question 9

True or false? The SSU and LSU of ribosomes contain basically the same set of proteins.

Answer 9

False, the SSU and LSU contains different sets of proteins

Question 10

What does an assembly map of the 30S ribosomal subunit show?

Answer 10

How a 30S ribosomal subunit of E. coli can be reassembled in the test tube from RNA and proteins, order of addition is important. Arrows indicate dependence in binding

Question 11

What are the two structural features of the Small ribosomal subunit?

Answer 11

• Head

- Base

Question 12

What are the four structural features of the large ribosomal subunit?

Answer 12

• Ridge
• Valley
• Central protuberance
• Stalk

Question 13

What three research methods were able to discern the secondary structure of 16S rRNA?

Answer 13

• Chemical and enzymatic probing
• Cross-linking studies
• Comparative sequence analysis (phylogenetic approach)

Question 14

Describe the secondary structure of E.coli’s 16S rRNA

Answer 14

• Short range base pairing generating characteristic helices in various parts of the structure.
• THere are long range base pairing regions that define four major folding domains (I-IV)

Question 15

When homologous sequences (ie. sequences that share a common ancestor) are seen in the primary structure of ribosomal subunits, what does this indicate?

Answer 15

That these are anchor regions that align sequences

Question 16

Comparative sequence analysis (phylogenetic approach) to discerning the secondary structure of ribosomal subunits looked for what two things?

Answer 16

• Primary sequence identities (homologous anchors)

- Compensating base changes in putative helical regions of the secondary structure

Question 17

What is the universal core in ribosomes?

Answer 17

A region of the SSU and LSU (separate in the two units) that consists of non-contiguous (not immediately adjacent) segments of primary sequence that interact through complementary base pairing to form long range helices which divide the secondary structure into discrete folding domains (I, II, III and IV)

Question 18

What is evidence for the universal core being essential to the function of ribosomes?

Answer 18

It is highly conserved in both primary and secondary structures in different organisms and organelles.

Question 19

What are two points of evidence for RNA having a direct role in protein synthesis and what is the conclusion of them (concerning the universal core)?

Answer 19

• LSU and SSU contains a highly conserved core of primary and secondary structure (universal core)
• When mRNA and tRNA interact, specific nucleotides in functionally important regions of these RNAs can be chemically cross linked to selected universal core nucleotides in SSU and LSU rRNAs

This means that these critical mRNAs and tRNA nucleotides are positions within and probably interact with the universal core region of rRNA

Question 20

In addition to evidence about the universal core of rRNA having a direct role in protein synthesis, what is further evidence that ribosomal RNA has a direct role in protein synthesis before x-ray crystallography? (3)

Answer 20

• Some antibiotics that inhibit protein synthesis bind to specific core nucleotides
• Mutations in universal core nucleotides make ribosomes resistant to antibiotics that normally bind to these nucleotides
• Large ribosomal subunits that have been stripped of protein are still able to carry out peptide bond synthesis (although complete deproteinization destroys this activity)

Question 21

What evidence was the final proof needed for RNAs functional importance in ribosomes?

Answer 21

x-ray crystallography proved that the active site is within the universal core and entirely surrounded by rRNA, not proteins.

Question 22

What are the tRNA binding sites on the ribosome? What subunit are they in?

Answer 22

E (empty)
P (peptidyl)
A (aminoacyl)

In the large subunit (LSU)

Question 23

What came first for the ribosome, RNA or protein?

Answer 23

Original ribosome consisted only on rRNA, ribosomal proteins came later

Question 24

What is the function of proteins in ribosomes?

Answer 24

To provide scaffold support and to stabilize the tertiary structure of rRNA in its catalytically active conformation

Question 25

How does the ribosome accelerate peptide-bond formation? (3)

Answer 25

- Positioning substrates - Reorganizing water in the active site - Providing electrostatic network that stabilizes reaction intermediates

Question 26

Describe proton transfer during peptide bond formation

Answer 26

Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate

Question 27

How is rRNA produced?

Answer 27

rDNA units tandemly linked together (one after another) and transcribed in the nucleolus. Along an rDNA repeat unit a 'christmas tree' looking structure appears. Each primary transcript comes out of a processome.

Question 28

What are the components of a primary ribosomal (rRNA) transcript? How is it modified?

Answer 28

- Mature rRNA - Spacers - The RNA is modified with 2'-O-methyls and pseudouridines - Spacer sequences are removed by the action of exo and endoribonucleases - Mature rRNAs are folded and assembled with ribosomal proteins All modifications happen at the same time

Question 29

What are the two types of spacers in ribosomal (rRNA) primary transcript?

Answer 29

External transcribed spacer (ETS) Internal transcribed spacer (ITS)

Question 30

How are ribosomes made in eukaryotes (ribosome biogenesis)? Starting with the ribosomal (rRNA) primary transcript

Answer 30

1. The 45S rRNA transcript is packaged into a large ribonucleoprotein particle (RNP) in the nucleolus, containing many ribosomal proteins imported from cytoplasm. 2. Sites in the rRNA are modified and spacer sequences in the rRNA are discarded 3. Large and small ribosomal subunits are exported from the nucleus individually

Question 31

What type of rRNA modifications take place in eubacteria?

Answer 31

Only a few sites of pseudouridine and 2-O'-methylation (Nm) occur Unique proteins catalyze modifications at each of these sites

Question 32

What type of rRNA modifications take place in eukaryotes and archaea?

Answer 32

many more sites of rRNA modifications occur in eukaryotic and archaeal rRNA than in eubacteria

Question 33

What is the significance to rRNA modifications?

Answer 33

Modified residues are positioned close to the ribosome active site, the peptidyl transferase centre (PTC), suggesting they contribute to structure/function