Green 13 - Riboswitches

Question 1

What are the functions of riboswitches?

Answer 1

The binding of small ligands (metabolites) to riboswitches cause a change in their structure which alters the expression of that particular metabolite
No regulatory protein intermediate is needed

Question 2

Where are riboswitches usually located?

Answer 2

In the 5’ UTR of mRNA

Question 3

Describe the 3 domains that exist w/in riboswitches.

Answer 3

Aptamer - binding of metabolite
Switching sequence - transmits the conformational change between aptamer and EP
Expression platform - can exist in one of 2 mutually exclusive conformations depending upon ligand binding. (anti)terminator loop

Question 4

Describe transcriptional attenuation (by using examples) as a mechanism for riboswitches to alter prokaryotic gene expression.

Answer 4

Transcriptional attenuation - as mRNA produced, formation of an anti/terminator loop determines whether transcription proceeds.
eg 1 - SAM - I riboswitch
- SAM = S-adenosyl methionine (precursor for Cys/Met synthesis)
- SAM - I riboswitch found upstream of genes involved I C/M synthesis in gram +ve bacteria
- low SAM = no SAM bound to riboswitch therefore antiterminator loop therefore no repression of transcription & it proceeds
- high SAM = SAM bound, change in secondary structure, formation of terminator loop, transcription halts when terminator loop makes contact w/ RNAP

eg 2 - T box riboswitch for B. subtilis Trp synthesis

• measures amounts of unloaded tRNAs as a way to see how much aa is present
• high Trp = loaded tRNAs w/ Trp, tRNA binds to specifier loop on mRNA but cannot bind attenuator because Trp blocks it, terminator loop
• low Trp = unloaded tRNAs, tRNA binds at both specifier loop & attenuator, binding @ attenuator stabilises the formation of the anti terminator loop therefore transcription of biosynthetic genes for Trp synthesis occurs

Question 5

What does the occlusion of the SDS target?

Answer 5

Because the SDS is where ribosomes bind, it targets translation initiation

Question 6

Give an example of an occlusion of the SDS mechanism.

Answer 6

Thymine pyrophosphate riboswitch

• TPP is a cofactor for C metabolism
• low TPP = no TPP bound, antiSD -> antiantiSD therefore SDS accessible by ribosomes and translation occurs
• high TPP = TPP bound, antiSD base pairs (intramolecularly) to the SD therefore not accessible by ribosomes therefore no translation.

The TPP switch controls the genes involved in thiamine biosynthesis (TPP is the active form of thiamine)

Question 7

What are ribozymes?

Answer 7

Ribozymes cleave mRNA using self-catalysis

Question 8

Give an example of a ribozyme

Answer 8

GlmS ribozyme

• located in 5’ UTR of the glmS gene
• GlmS enzyme is needed for the conversion of fructose & glutamine -> glucosamine-6-P (GlcN6P)
• low GlmS = no GlmS binds to the transcribed GmlS mRNA therefore translation proceeds as normal
• high GlmS = GlmS binds to mRNA, conformational change into active ribozyme which cleaves its own mRNA, this cleaved mRNA is then directed for more degradation by ribonuclease, no translation, no GlmS enzyme and no GlcN6P production