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| Status |
Public on Sep 30, 2024 |
| Title |
Optimized periphery-core interface increases fitness of the Bacillus subtilis glmS ribozyme |
| Organism |
Bacillus subtilis |
| Experiment type |
Other
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| Summary |
Like other functional RNAs, ribozymes contain a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-peripheral interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (ClvSeq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. Most deleterious mutations impaired RNA self-assembly. All-atom MD simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop rewire the network of hydrogen bonds around the catalytic site. Remarkably, IL4 mutations introduce a non-native helix interface that corrupts folding of the wild type core, eliminating activity. The results illustrate how competition between native and non-native structures in RNA drives the natural selection of central and peripheral tertiary interaction motifs.
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| Overall design |
The plasmid variant library was in vitro transcribed, allowed to self-cleave in the presence of Mg2+ and GlcN6P, purified by PAGE and reverse transcribed into 12 barcoded output cDNA sub-libraries that were pooled and amplified for paired-end HiSeq.
Two barcoded versions of the WT ribozyme were added to the purified RNA pools as spike-in controls to estimate absolute RNA concentration.
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| Contributor(s) |
Yu D, Woodson S |
| Citation missing |
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| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R35 GM136351 |
Assembly Mechanisms of RNA-Protein Complexes for Genetic Control |
JOHNS HOPKINS UNIVERSITY |
Sarah A. Woodson |
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| Submission date |
Mar 11, 2024 |
| Last update date |
Sep 30, 2024 |
| Contact name |
Sarah A Woodson |
| E-mail(s) |
swoodson@jhu.edu
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| Organization name |
Johns Hopkins University
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| Department |
Biophysics
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| Street address |
3400 N. Charles St.
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21218 |
| Country |
USA |
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| Platforms (1) |
| GPL29318 |
Illumina HiSeq 3000 (Bacillus subtilis) |
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| Samples (12)
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| GSM8141347 |
glmS ribozyme variant library, un-cleaved, 0 s |
| GSM8141348 |
glmS ribozyme variant library, un-cleaved, 5 s |
| GSM8141349 |
glmS ribozyme variant library, un-cleaved, 10 s |
| GSM8141350 |
glmS ribozyme variant library, un-cleaved, 30 s |
| GSM8141351 |
glmS ribozyme variant library, un-cleaved, 60 s |
| GSM8141352 |
glmS ribozyme variant library, un-cleaved, 180 s |
| GSM8141353 |
glmS ribozyme variant library, cleaved, 0 s |
| GSM8141354 |
glmS ribozyme variant library, cleaved, 5 s |
| GSM8141355 |
glmS ribozyme variant library, cleaved, 10 s |
| GSM8141356 |
glmS ribozyme variant library, cleaved, 30 s |
| GSM8141357 |
glmS ribozyme variant library, cleaved, 60 s |
| GSM8141358 |
glmS ribozyme variant library, cleaved, 180 s |
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| Relations |
| BioProject |
PRJNA1086553 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE261357_gS_mut_zone_ref.txt.gz |
119 b |
(ftp)(http) |
TXT |
| GSE261357_gS_sm_FitParam.txt.gz |
3.9 Kb |
(ftp)(http) |
TXT |
| GSE261357_gS_sm_raw_counts.txt.gz |
27.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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