The Folding Kinetics of Riboswitch Transcriptional Terminators

1
The Folding Kinetics of Riboswitch
Transcriptional Terminators

• Mike Widom
• Ben Sauerwine
• Carnegie-Mellon University
• RNA Club
• October 10, 2008

2
What is a Riboswitch?
Figure from Wikipedia article Transcription
(genetics)

• Figure by Wade C. Winkler, Smadar
  Cohen-Chalamish, and Ronald R. Breaker, An mRNA
  structure that controls gene expression by
  binding FMN, PNAS December 10, 2002 vol. 99 no.
  25 15908-15913

3
Test Cases
Are biological terminator hairpins selected for
efficiency in their folding pathways?

• Purine Riboswitches
• FMN Riboswitches
• Bacillus Subtilis
• Streptococcus Family

4
Folding Methods

• ViennaRNA package
• RNAfold MFE structures
• Kinfold Monte Carlo Simulations
• 1 Kinfold step 5 microseconds

5
Controls

• Reverse Growth
• Shuffled Sequences

6
Average Formation vs. Inverse Growth Rate
Bacterial Growth Rate
yxjB
50 nt/s
Faster
Slower
7
Purine in Bacillus Subtilis
yxjB
ydhL
xpt
Average Proportion MFE Found
pbuG
purE
ydhL
Grow Time (Kinfold Units 4000 corresponds to 50
nt/s)
8
FMN in Bacillus Subtilis
MtrB
Average Proportion MFE Found
ypaA
Grow Time (Kinfold Units 4000 corresponds to 50
nt/s)
9
Purine in Streptococcus Family
Unknown gene
xpt
Average Proportion MFE Found
Unknown gene
xpt
Grow Time (Kinfold Units 4000 corresponds to 50
nt/s)
10
FMN in Streptococcus Family
ribD
ribD
spr0433
Average Proportion MFE Found
sag1403
Unknown gene
Unknown gene
Grow Time (Kinfold Units 4000 corresponds to 50
nt/s)
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Distribution of Trials
12
Distribution of Average MFE Structure Found
Real Biological Sequence
13
Purine in Bacillus Subtilis
yxjB
ydhL
xpt
Counts
pbuG
purE
ydhL
Average Proportion MFE Found
14
Purine in Streptococcus
Unknown gene
xpt
Counts
Unknown gene
xpt
Average Proportion MFE Found
15
FMN in Bacillus Subtilis
MtrB
Counts
ypaA
Average Proportion MFE Found
16
FMN in Streptococcus
ribD
ribD
spr0433
Counts
Unknown gene
sag1403
Unknown gene
Average Proportion MFE Found
17
Significance
18
Significance
19
How do Transcriptional Terminators fail?
purE grown normally
purE grown from 3 to 5 end
20
Is Nucleotide Distribution a Factor?
21
Conclusions

• Inefficient folding pathways are negatively
  selected in riboswitch terminator hairpins.
• Kinetics as well as thermodynamics is important
  for performance of a biological riboswitch.