Gene regulation by riboswitches

1
Gene regulation by riboswitches
1. Regulation of gene expression 2. Principles
and examples of RNA-mediated genetic control 3.
Riboswitches - Organization and properties -
Transcription termination (FMN riboswitch) -
Translation inhibition (thiamine riboswitch) -
RNA cleavage (glmS riboswitch) 4. Summary
2
Control of gene expression
mRNA degradation

• protein degradation
• export

• protein synthesis
• protein folding

• mRNA synthesis
• mRNA folding

Transcription
Translation
3
Principles of RNA-mediated genetic control (I)
Transcription termination
OFF
ON
Repression
Activation
4
Principles of RNA-mediated genetic control (II)
Translation initiation
ON
OFF
Repression
Activation
5
Signals of RNA-mediated genetic control
OFF
ON
Repression
Activation
Ribosome
Protein
RNA
Metabolite
Temperature
6
Example 1 Ribosome - Transcription attenuation
trp genes of E. coli
7
Example 2 RNA-binding protein
ptsGHI genes of B. subtilis
antiterminator
8
Example 3 Antisense RNA
rpoS gene of E. coli
9
Example 4 tRNA
glyQS genes of B. subtilis
10
Example 5 Temperature
prfA gene of L. monocytogenes
11
Example 6 Metabolite (Riboswitch)
thi operon of B. subtilis
12
Organization and properties of riboswitches (I)

• conserved genetic control elements in the 5
  untranslated region (UTR) of bacterial mRNAs
• are regulated by metabolites coenzymes (B1, B2,
  B12), amino acids, purin bases
• consist of only RNA
• estimation gt 2 of all genes are regulated by
  riboswitches

13
Organization and properties of riboswitches (II)

• structured binding pocket
• high affinity
• high specificity

Binding domain (Aptamer)

• reacts upon ligand binding by the aptamer
• comformational change alters gene expression

Regulatory Domain (Expression platform)
14
Regulation of riboflavin biosynthesis in B.
subtilis

• RFN element in mRNAs of genes required for
  biosynthesis of riboflavin and FMN
• FMN required for repression of the ribDEAHT
  operon of B. subtilis
• mutations in the RFN element eliminate
  FMN-mediated repression of the ribDEAHT operon

15
Spontaneous hydrolysis of RNA
16
RNA analysis by in-line probing
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The RFN element is a riboswitch
Winkler et al. (2002), PNAS 99, 15908-13 Mironov
et al. (2002), Cell 111, 747-756
ribD mRNA
18
The RFN element is a FMN riboswitch
Winkler et al. (2002), PNAS 99, 15908-13
3 ?M
apparent KD
5 nM
300 nM
19
The FMN riboswitch terminates transcription
Winkler et al. (2002), PNAS 99, 15908-13
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Termination of transcription

• RNAP/DNA/RNA complex (TEC) is exceptionally
  stable
• Transcription is processive RNAP does not come
  off the DNA until termination

pause site 11 bases from hairpin
downstream DNA
UUUUUUUU

• hairpin inactivates and destabilizes TEC
• Us destabilize hybrid

21
Single-round transcription (synchronized)
Phillip et al. (2001), Methods Enzymol 340, 466-85
displaces RNAP from nonspecific binding sites
?-32PUTP, GTP, ATP
nascent RNA paused at the first G of template
strand
22
How does the FMN riboswitch really work?
Wickiser et al. (2005), Molecular Cell 18, 49-60
Single round transcription assay

• apparent dissociation constant KD ? 5 nM
• conc. to induce 50 termination ? 500 nM

Does transcription kinetics operate the FMN
riboswitch?
23
Role of NusA in transcription termination
Borukhov et al. (2005), Mol. Microbiol. 55,
1315-1324

• essential transcription elongation factor
  (conserved among eubacteria and archaea)
• stimulates pausing
• can induce anti-termination

NusA-induced RNAP pausing provides a mechanism
for synchronizing transcription and translation
24
Role of NusA in transcription termination
Borukhov et al. (2005), Mol. Microbiol. 55,
1315-1324
25
NusA reduces the effective FMN concentration
Wickiser et al. (2005), Molecular Cell 18, 49-60
Single round transcription assay
NusA increases the fraction of terminated
transcripts by 2-fold
26
Identification of transcriptional intermediates
Wickiser et al. (2005), Molecular Cell 18, 49-60
Single round transcription assay
27
Life time of pause sites
Wickiser et al. (2005), Molecular Cell 18, 49-60
Single round transcription assay
60 sec
10 sec

• NusA increases lifetimes
• increasing concentrations of NTPs descreases
  lifetimes

28
Dependence of ligand affinity on transcript length
Wickiser et al. (2005), Molecular Cell 18, 49-60
PB mimic
PA mimic
aptamer
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Binding of FMN by transcriptional intermediates
Wickiser et al. (2005), Molecular Cell 18, 49-60
PB mimic
aptamer
FMN binding
FMN binding (165 ribD and 230 ribD)
antiterminator (244 ribD)
10 nM
apparent KD
100 nM
-
30
Binding of FMN by transcriptional intermediates
Wickiser et al. (2005), Molecular Cell 18, 49-60
Fluorescence quenching
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Binding of FMN by transcriptional intermediates
Wickiser et al. (2005), Molecular Cell 18, 49-60
Fluorescence quenching
PA mimic
aptamer
PB mimic
32
Binding kinetics of FMN to its aptamers
Wickiser et al. (2005), Molecular Cell 18, 49-60
Time-resolved fluorescence quenching
M-1s-1
Estimation of times for binding of 50 of ribD
mRNA by FMN (1 ?M FMN excess over mRNA, 37 ?C)
2 s for 200 ribD 34 s for 230 ribD
33
Life time of pause sites
Wickiser et al. (2005), Molecular Cell 18, 49-60
Synchronized transcription assay
60 sec
10 sec

• substantial amount of has reached pause sites
  within 8 s
• majority of mRNAs have reached termination site
  after 60 s

Without the pause sites much more than 1 ?M FMN
would be needed to bind the riboswitch with
sufficient speed.
34
Transcriptional intermediates - Conclusions
Wickiser et al. (2005), Molecular Cell 18, 49-60

• Aptamer remains receptive to FMN binding until
  it is disrupted by the formation of the
  antiterminator helix. irreversible event of the
  riboswitch at low FMN concentrations
• longer transcripts have lower affinity
• aptamers of transcripts paused at PA and PB have
  affinities better than the T50 values

35
Life time of the RNA-FMN complex
Wickiser et al. (2005), Molecular Cell 18, 49-60
In vitro transcription observed by fluorescence
quenching
FMN dissociates only very slowly from the ribD
mRNA.
36
Life time of the RNA-FMN complex
Wickiser et al. (2005), Molecular Cell 18, 49-60
Dissociation kinetics of the 165 ribD/FMN complex
(100 nM, 25 ?C)
koff 10-3 s-1 ? k ? 10 min
37
Kinetics of antiterminator helix formation
Wickiser et al. (2005), Molecular Cell 18, 49-60
38
FMN and the antiterminator bind competitively to
ribD mRNA
Wickiser et al. (2005), Molecular Cell 18, 49-60
Dissociation kinetics of the 230 ribD/FMN complex
(150 nM, 25 ?C)
antiterminator

• 10-fold excess of ATO over FMN reduces ribD/FMN
  association by half
• kon 103 M-1s-1 100-fold slower than typical
  nucleic acid hybridization times
• comparison with other intramolecular helix
  formations gt 3 s for antiterminator formation

39
Kinetic model for the FMN riboswitch
Wickiser et al. (2005), Molecular Cell 18, 49-60
1 ?M FMN, 25 ?C

• control by kinetic competition coupling of
  regulatory reaction to rate of RNA synthesis
• FMN binding occurs primarily while RNA
  polymerase is paused
• Caution - gt half of the in vitro transcripts
  do not function as predicted -
  low NTP concentration thermodynamic control

40
Regulation of thiamine biosynthesis genes in E.
coli
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The thi box is a riboswitch
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The TPP riboswitch inhibits translation initiation
43
Structure of the thiamine pyrophosphate riboswitch
Serganov et al. (2006), Nature 441, 1167-1171
44
Cleavage of RNA by a riboswitch
Winkler and Dann (2006), Nat Struct Mol Biol 13,
569-71
45
Structure of the glmS riboswitch
Klein and Ferre-D'Amare (2006), Science 313,
1752-6
46
The glmS riboswitch has pre-formed coenzyme
binding pocket
Klein and Ferre-D'Amare (2006), Science 313,
1752-6
47
Proposed catalytic mechanism of the glmS ribozyme
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Summary

• regulatory elements which control a wide set of
  metabolic pathways in bacteria
• consist solely of RNA
• Aptamer domain high affinity and specific
  binding of metabolites
• Expression platform regulation of gene
  expression by control of
• - transcription termination kinetically
  controlled
• - translation initiation
• - RNA stability