Lecture 15: Bacterial Translation

Question 1

What is the meaning of polycistronic?

Answer 1

Can encode multiple genes

Question 2

Where are ribosome binding sites located?

Answer 2

Upstream of the gene

Question 3

Is a ribosome binding site needed for each gene?

Answer 3

Yes

Question 4

Is it true that many ribsomes can travel along a mRNA molecule at the same time?

Answer 4

Yes

Question 5

What 2 subunits are bacterial ribosomes made up of?

Answer 5

50S and 30S

Question 6

What is the 50S ribsosmal subunit made up of?

Answer 6

23S rRNA + 5S rRNA + 31 proteins

Question 7

What does the large 50S ribosomal subunit do?

Answer 7

It catalyses the formation of peptide bonds that join amino acids together.

Question 8

What does the small 30S ribosomal subunit do?

Answer 8

It provides a framework that matches tRNAs to codons

Question 9

What is the 30S ribosomal subunit made up of?

Answer 9

16S rRNA + 21 proteins

Question 10

What is a Svedberg unit (S)?

Answer 10

A unit for sedimentation coefficient. It is a measure of size based on sedimentation rate, i.e. how fast a particle of given size and shape ‘settles’ to the bottom of a solution.

Question 11

Are Svedberg units additive?

Answer 11

No

Question 12

What ribsome does bacterial translation occur on?

Answer 12

The 70S ribosome

Question 13

What do ribosomal proteins do to the ribsosme?

Answer 13

Ribosomal proteins stabilise the ribosome

Question 14

How do ribosomal proteins stabilise the ribosome?

Answer 14

Because they have unstructured extensions that snake into the RNA framework, stabilising the ribosome.

Question 15

How does the ribosome find the mRNA?

Answer 15

The 16S rRNA (30S subunit) of the ribsosome binds to the Shine-Dalgarno sequence. This recruits the ribosome

Question 16

where is the Shine-Dalgarno sequence located?

Answer 16

A few bases upstream of the start codon. The sequence is dominated by purines.

Question 17

Describe the initiation step of bacterial translation

Answer 17

• The 30S subunit associates with the initiation factors. It binds to IF-1 and IF-3. Binding to IF-1 blocks the A site, so prevents tRNAs from prematurely binding to the A site. Binding to IF-3 prevents premature assembly of the ribosomal S and L subunits until both mRNA and initiating tRNA are available.
• The S subunit then binds to the Shine-Dalgarno sequence, as he Shine-Dalgarno sequence is complementary to a region on the 16S rRNA of the 30S subunit. The mRNA is then aligned so that the start codon (AUG) is in the P site.
• IF-2 (which is bound to GTP) then guides the initiating tRNA to the P site/start codon.
• Once the start codon is recognised, the large subunit is then recruited to the 30S complex and the initiation factors (IF-1 and IF-2 ) are released.
• The fully assembles ribosome (70S) is now ready for elongation, the P site is occupied by the initiating tRNA, and the A and E sites are empty.

Question 18

Describe the elongation step of bacterial translation

Answer 18

• The ribosome reads the next codon in the mRNA located in the A site.
• EF-Tu (which is bound to GTP) guides the next amino acid from the P site to the acceptor site
• Ef-Tu then dissociates and diffuses away, and t ribosome’s peptidyl transferase center catalyses the formation of a peptide bond between the 2 adjacent amino acids.
• EF-G (which is bound to GTP) is a translocase that enters the A site. It pushes the ribosome one codon down towards the 3’ end of the mRNA.
• The deacylated tRNA enters the E site and the dipeptidyl tRNA enters the P site.
• An incoming amino acid tRNA then displaces EF-G , a peptide bind is formed between the 2 former amino acids, and the uncharged tRNA in the E site leaves.
• The cycle then repeats again and again till a stop codon is reached so that no more tRNA binds, and the mRNA ribosome assocaition is broken.

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Question 19

In the ribosome, what’s the A site?

Answer 19

(Aminoacyl site): Where incoming charged tRNAs bind.

Question 20

In the ribosome, what’s the P site?

Answer 20

(Peptidyl site): Holds the tRNA carrying the growing polypeptide chain.

Question 21

In the ribosome, what’s the E site?

Answer 21

(Exit site): Where uncharged tRNAs exit the ribosome.

Question 22

What do charged tRNAs do?

Answer 22

They carry amino acids

Question 23

What are the EFs?

Answer 23

The elongation factors

Question 24

What does EF-Tu do?

Answer 24

It brings aminoacyl-tRNA to the A site in a GTP-dependent manner.

Question 25

What does EF-Ts do?

Answer 25

It regenrates EF-Tu by exchanging GDP for GTP.

Question 26

What does EF-G do?

Answer 26

It facilitates ribosome translocation.

Question 27

What does the Peptidyl Transferase Center (PTC) do?

Answer 27

It catalyses peptide bond formation. (part of the 23S rRNA in the 50S ribosomal subunit).

Question 28

What does GTP provide energy for?

Answer 28

The elongation steps

Question 29

What is head polymerisation?

Answer 29

Where the growing amino acid is transferred to the next amino acid that’s brought in

Question 30

What is required in order for a peptide bond to be formed between 2 amino acids?

Answer 30

Peptidyl transferase activity, which is provided by the 23S rRNA of the L subunit.

Question 31

Describe bacaterial termination

Answer 31

• A release factor (rf) binds to the stop codon
• The peptidyl-tRNA bond is cleaved, releasing the protein
• The mRNA-ribosome complex dissasembles

Question 32

What is the difference between bacterial and prokaryotic ribsosomes?

Answer 32

• Bacteria have a 70S ribosome, whilst eukaryotes have an 80S ribosome
• Eukaryotic ribosomes are bigger
• Eukaryotic ribosomes don’t have a clearly defined E site

Question 33

Describe the intiation step of eukaryotic translation

Answer 33

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Question 34

Is the mRNA in eukaryotes capped at the 5’ end?

Answer 34

Yes.

Question 35

What replaces the Shine-Dalgarno sequence in eukaryotes?

Answer 35

The Kozak sequence, this facilitates intiation.

Question 36

What does the fact that eukaryotic mRNAs are pseudo-circularised mean?

Answer 36

the 5’ and 3’ ends are bound by specific proteins that interact, facilitating the re-binding of ribosomes. This increases the efficiency of re-initiation