Translation 3- Regulation

Question 1

How does the B. subtilis tenA riboswitch regulated?

Answer 1

By binding of TPP. When TPP concentration is low, a 2/3 stem loop forms and allows transcription of downstream genes.

Question 2

How is the Vibrio cholerae riboswitch in the 1422 UTR regulated?

Answer 2

By binding glycine. When no Gly is present, 2/3 stem loop forms and acts as a terminator and blocks RBS. When Gly is present, 1/2 stem loop forms and allows ribosome access.

Question 3

How do small regulatory RNAs regulate translation in bacteria?

Answer 3

Associate with Hfq, can then bind to RBS to regulate translation initiation or to an mRNA to regulate degradation by recruiting an RNase.

Question 4

What is the stringent response?

Answer 4

Causes a coordinated shutdown of mRNA, tRNA and rRNA synthesis during nutrient starvation. Ribosome becomes blocked by uncharged tRNA due to aas not being available. Blocked ribosome is detected by RelA which catalysts pppGpp synthesis.

Question 5

What does RelA catalyse?

Answer 5

Catalyses pppGpp synthesis from ATP and GTP

Question 6

What catalysts the conversion of pppGpp to ppGpp?

Answer 6

GPPA

Question 7

What is catalysed by SpoT?

Answer 7

The breakdown of ppGpp into GDP and pyrophosphate.

Question 8

What processes are shutdown by the stringent response?

Answer 8

Transcription, translation, replication initiation, cell division and active transport.

Question 9

What processes are stimulated by the stringent response?

Answer 9

Amino acid biosynthesis and the production of the sigma factor used in nutrient starvation- upregulates stress and starvation genes.

Question 10

How does ppGpp inhibit translation?

Answer 10

Binds to the omega/beta’ interface of RNAP, reducing RNAP activity.

Question 11

What happens after RelA has catalysed pppGpp synthesis?

Answer 11

It is released from the ribosome, and can go on to detect more blocked ribosomes.

Question 12

What happens at the end of starvation?

Answer 12

The concentration of ppGpp must decrease for growth to continue. Failure to breakdown ppGpp (SpoT) can result in cell death.

Question 13

How is translation resumed following the end of nutrient starvation?

Answer 13

Aminoacylated tRNAs displace the uncharged tRNAs in the A site of the ribosome.

Question 14

How does ribosome dimerisation regulate translation during starvation and stress?

Answer 14

70S ribosomes bind RMF to form 90S dimers. Dimers bind HPF to form 100S ribosomal particles. These dimers are translationally inactive.

Question 15

How does the RBS/Shine Dalgarno regulate translation?

Answer 15

lower sequence homology to other SD sequences, lower rate of translation initiation. Allows different amounts of different proteins to be produced (when the genes are in a polycistronic mRNA).

Question 16

How does mRNA capping regulate translation in eukaryotes?

Answer 16

Prevents degradation of mRNA by 5’ exonuclease. Initiation of translation is cap dependent. Ribosome is positioned via interaction with the cap.

Question 17

How does polyadenylation regulate translation in eukaryotes?

Answer 17

Poly(A) binding protein prevents 3’ degradation of mRNA. Ribosome positioning occurs via interaction with the poly(A) tail.

Question 18

What is the ramp theory?

Answer 18

The order of high/low efficiency codons controls the translation efficiency and reduces ribosome traffic. Second codon is translated rapidly to allow recycling of the initiator tRNA. First 50 codons are rarer, slowing elongation.

Question 19

Where are uORFs found?

Answer 19

Growth and development genes in vertebrates. Found in 2/3 of oncogenes.

Question 20

What is the role of uORFs?

Answer 20

To regulate expression and translation of a downstream major ORF.

Question 21

What are riboswitches?

Answer 21

Secondary structures that form within mRNA and regulate translation by blocking RBS access. Riboswitches are regulated by small aptamers (often metabolites, e.g. Gly)- this affects the secondary structure that forms. ‘Acts as the switch’

Question 22

How does YfiA affect ribosome dimerisation?

Answer 22

Structurally similar to HPF. Binds to ribosomes when in stationary phase in E.coli. Binds to A/P sites.

Question 23

What does RsfS do?

Answer 23

Can block the binding of two ribosomal subunits, inhibiting translation.

Question 24

Describe mRNA masking in eukaryotes.

Answer 24

mRNA is bound by a ribonucleoprotein that prevents association with the ribosome. Proteins dissociate from mRNA after a phosphorylation or dephosphorylation signal. Regulates translation and protein synthesis.

Question 25

When is mRNA masking often used to regulate protein synthesis?

Answer 25

During early embryonic development.

Question 26

How does antisense RNA regulate protein synthesis in eukaryotes?

Answer 26

Forms dsRNA with complementary mRNA- this cannot be translated by the ribosome.

Question 27

How do interferons prevent further viral infection?

Answer 27

Are secreted by virus infected cells and inhibit protein synthesis in infected cells- prevents formation of viral proteins.

Question 28

Name the mRNA degradation pathways.

Answer 28

Capped and Decapped degradation.

Question 29

Describe the capped mRNA degradation pathway.

Answer 29

Poly(A) tail removed first. Once only 15 residues in length, poly(A) binding protein can no longer bind. Allows 3’ to 5’ exonuclease degradation. Can have decapping and degradation from the opposite end,

Question 30

Describe the decapping mRNA degradation pathway.

Answer 30

5’ cap is removed first. Exposes mRNA to an extremely aggressive 5’ to 3’ exonuclease. (Not degraded by 3-5 due to aggressiveness of 5-3)

Question 31

How does RNA capping occur?

Answer 31

1. When the C-terminal domain of RNAP is phosphorylated, it recruits the capping enzyme complex. 2. Guanylyl-transferase removes the γ-phosphate of the 5’ nucleotide and the β and γ phosphates of GTP- adds a guanosine to the 5’ end of mRNA via a 5’ to 5’ triphosphate linkage. 3. Guanine methyltransferase converts the added guanosine to 7-methylguanosine.

Question 32

How does polyadenylation occur?

Answer 32

1. Poly(A) hexanucleotide signal binds to the Cleavage and Polyadenylation Specifity factor and the GU-rich downstream element binds to the Cleavage Stimulation factor, both on the mRNA.2. This positions the mRNA 3’end for cleavage of the GU-rich region at the 3’ end of pre-mRNA, by an endonuclease. 3. Poly(A) polymerase produces the poly(A) tail, using ATP. 4. Poly(A) binding protein binds.

Question 33

Does poly(A) polymerase require a template to synthesise the poly(A) tail?

Answer 33

No