Lecture 7 What happens after transcription initiation 2 Flashcards
What are the three ways by which attenuation can be controlled?
- RNA binding protein
- ribosome
- riboswitch
What is a riboswitch?
- 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
What are the two functional domains of the RNA of a riboswitch?
- Aptamer - a metabolite sensing domain
- Expression platform - e.g. SD sequence
Metabolite interacts with the aptamer
Where are riboswitches found?
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)
What is the generic simple riboswitch?
- promotion of specific folding pathways by metabolite binding, allows specific structures to form and prevent others from forming
- changes structure at the active site
What can changes in gene expression can riboswitches result in?
- affect transcript cleavage (via ribozyme - splicing)
- positively affect transcription by disruption the terminator formation
What elements of transcription/translation can the riboswitch regulate?
- transcription elongation
- translation initation
- RNA processing
How do riboswitches mostly act in bacteria?
Negitively
- terminate transcription
- inhibit translation initiation
How can riboswitches terminate transcription?
- 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
How can riboswitches inhibit translation initiation?
- 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)
What experimental approaches can be used to study a riboswitch and/or confirm regulation by a riboswitch?
- 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
How can riboswitches be experimentally tested through examining gene expression in vivo with/without a ligand?
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
What mechanism of riboswitch is present in purine biosynthesis genes?
-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
What mechanism of riboswitch are the SAM riboswitches?
- two different riboswitches working with the same metabolites
- SAM= S-adenosyl methionine (universal methyl group donor)
- SAM riboswitch I and II
- gram positive bacteria
What 2 bacterial species have a conserved sequence in elements of structural importance in the SAM binding motif?
E.faecalis and S.gordonii
What would be the outcome of a β-galactosidase assay on the purine biosynthesis gene riboswitch?
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
What are the features of the SAM riboswitch I?
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
What are the features of the SAM riboswitch II?
- Attenuation acts negatively by inhibiting translation initiation
- mechanism varies between species
- RBS occluded by stem and loop
Are there many or few ligands that can be involved in riboswitches?
-a diverse range of ligands can be involved
What is the history of discovery of riboswitches?
- 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)
How common are riboswitches in nature?
- In the Gr+ bacterium bascillus subtilis 2% of the genome are riboswitches
- TPP in eukaryotes
How can riboswitches/aptamers be used for designer bacteria?
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
Give an example of controlling protein activity where no factor/ligand is required for RNA structure change
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)
