Lecture 7 What happens after transcription initiation 2

Question 1

What are the three ways by which attenuation can be controlled?

Answer 1

• RNA binding protein
• ribosome
• riboswitch

Question 2

What is a riboswitch?

Answer 2

• A mechanism to regulate gene expression found in all three domains of life
• a cellular metabolite (not exclusively - can bind ions (Mg2+)/regulatory nucleotides (cl-dGMP)) interacts directly with regulatory elements within the untranslated regions of mRNA
• inducing structural changes that result in regulation of gene expression (after transcription) by:
• affecting transcription termination
• translation initiation
• RNA processing

Question 3

What are the two functional domains of the RNA of a riboswitch?

Answer 3

1. Aptamer - a metabolite sensing domain
2. Expression platform - e.g. SD sequence

Metabolite interacts with the aptamer

Question 4

Where are riboswitches found?

Answer 4

Prokaryotes
-5’ untranslated regions in mRNA
Eukaryotes
-untranslated region or within introns (can have a larger variety of function, RNA processing, RNA transport, expression pathways)

Question 5

What is the generic simple riboswitch?

Answer 5

• promotion of specific folding pathways by metabolite binding, allows specific structures to form and prevent others from forming
• changes structure at the active site

Question 6

What can changes in gene expression can riboswitches result in?

Answer 6

• affect transcript cleavage (via ribozyme - splicing)

- positively affect transcription by disruption the terminator formation

Question 7

What elements of transcription/translation can the riboswitch regulate?

Answer 7

• transcription elongation
• translation initation
• RNA processing

Question 8

How do riboswitches mostly act in bacteria?

Answer 8

Negitively

• terminate transcription
• inhibit translation initiation

Question 9

How can riboswitches terminate transcription?

Answer 9

• somewhere in RNA there is a potential terminator or anti terminator
• when the ligand interacts with the aptamer the terminator forms in the expression platform
• RNA pol stops transcribing and falls off

Question 10

How can riboswitches inhibit translation initiation?

Answer 10

• when the aptamer is unbound by the ligand the ribosome can bind to the expression platform and and translation can continue
• When the ligand binds to the aptamer a secondary stemp/loop structure forms in the expression platform (includes the RBS) and the RBS is blocked in the secondary structure (need ssRNA for translation)

Question 11

What experimental approaches can be used to study a riboswitch and/or confirm regulation by a riboswitch?

Answer 11

• Examine gene expression in vivo with/without a ligand
• Determine the structure of RNA with/without a ligand
• Bioformatics -> predict the riboswitch based on predicted structures of mRNA from genome sequences

Question 12

How can riboswitches be experimentally tested through examining gene expression in vivo with/without a ligand?

Answer 12

Reporter fusion
-detects if a metabolite controls transcription and expression of a specific gene
-use transcriptional or translational fusion
-with lacZ as a reporter (or GFP)
[lacZ from lac operon good as results can be easily quantified (blue/white colour changes when lacZ active) from the addition of a β-galactoside (lactose)]

Translational fusion

• Take region with the GOI SD sequence and make a protein fusion to lacZ so that all the information in translation and transcription comes from the GOI region
• visualise -> increase/decrease in lacZ expression could be due to regulation in the GOI region
• however not clear whether this is through promoter activity or translational activty

Transcriptional fusion

• insert lacZ to the region with the GOI with its own SD sequence but no promoter
• if there is a high concentration of lacZ this suggests that regulation occurred at the transcription level
• however don’t know if it occurs at the translational level too

Question 13

What mechanism of riboswitch is present in purine biosynthesis genes?

Answer 13

-simple
-guanine and adenine aptamers
Riboswitch shows:
-selectivity: 20,000X greater affinity for G with G aptamer than for A
-uses simple Watson-Crick base pairing
-single C to U mutation changes a G- to an A- recognising aptamer
-this is due to RNA secondary structure that the metabolite sits within

Question 14

What mechanism of riboswitch are the SAM riboswitches?

Answer 14

• two different riboswitches working with the same metabolites
• SAM= S-adenosyl methionine (universal methyl group donor)
• SAM riboswitch I and II
• gram positive bacteria

Question 15

What 2 bacterial species have a conserved sequence in elements of structural importance in the SAM binding motif?

Answer 15

E.faecalis and S.gordonii

Question 16

What would be the outcome of a β-galactosidase assay on the purine biosynthesis gene riboswitch?

Answer 16

Experiment
-Translational fusion and look at 2hours after
-Tested specific point mutations to identify the effect
W/O adenine - no difference (control)
W adenine - shows massive increase in expression
-> therefore regulation occurs at the translational level

If no difference was shown with adenine, then nothing would be happening at the regulational level

Question 17

What are the features of the SAM riboswitch I?

Answer 17

Attenuation acts negatively by premature termination of transcription elongation

• in on state, the antiterminator forms and the ribosome can bind to the RBS and transcribe the mRNA
• in the off state, SAM binding to the aptamer leads to the formation of the terminator stem/loop, occluding the RBS

Question 18

What are the features of the SAM riboswitch II?

Answer 18

• Attenuation acts negatively by inhibiting translation initiation
• mechanism varies between species
• RBS occluded by stem and loop

Question 19

Are there many or few ligands that can be involved in riboswitches?

Answer 19

-a diverse range of ligands can be involved

Question 20

What is the history of discovery of riboswitches?

Answer 20

• successful studies in the lab on biotechnological uses for aptamers found molecules that interact with RNA
• if work in lab, probably exist in nature. Naturally occurring riboswitches were found
• three groups worked on the idea in parallel [Henkin (attenuation background - manually identified conserved and interesting RNA structures in functional related enzymes), Breaker (biotech background - found in vtro RNA-ligand interactions), Nudler (transcription background - in vitro analysis of two regulatory mechanisms)

Question 21

How common are riboswitches in nature?

Answer 21

• In the Gr+ bacterium bascillus subtilis 2% of the genome are riboswitches
• TPP in eukaryotes

Question 22

How can riboswitches/aptamers be used for designer bacteria?

Answer 22

As sensors

• E.coli reprogrammed to move towards a compound of choice
  e. g. a synthetic theophyline sensing aptamer controlling the expression of a genes (cheZ) that regulates directed swimming in response to caffeine

How?

• made synthetic aptamer for caffeine and conjugated it to the protein necessary for motility (cheZ)
• plated cells with caffeine on one side and theophyline on the other
• WT showed no direction, whereas the synthetic did

As antibacterial
-find riboswitch controlling vir

Question 23

Give an example of controlling protein activity where no factor/ligand is required for RNA structure change

Answer 23

Temperature sensing

• anything with a secondary structure is sensitive to temperature
  e. g. RNA thermosensor in Listeria monocytogenes
• contains a G+ rod, food borne pathogen that can cause meningitis
• key regulator of vir is PrfA
• expression is thermoregulated (30C vs 37C)
  e. g. DNA structure curvature: protein-DNA interactions (e.coli)