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| Status |
Public on Mar 31, 2021 |
| Title |
The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis |
| Organism |
Escherichia coli |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity on the ribosome remain elusive. Here, we show a novel role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using random mutagenesis screening and functional studies, we identify a loop region (residues 114-130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wildtype RelA. Our data provide strong evidence to support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.
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| Overall design |
The relA locus of Escherichia coli was fused to a dual affinity His-TEV-FLAG tag. Strains expressing relA-HTF (RelA), relAA21E-HTF (A121E) were grown exponentially in MOPS minimal medium. Cell samples were UV-C irradiated (100sec of 1800mJ) before and 5 or 30 min after isoleucine starvation (indicated by 5 or 30) and harvested. Cells were washed with ice-cold PBS before snap freezing in liquid nitrogen. RNA-RelA complexes were purified on M2-anti-FLAG resin and Ni-NTA resin. Sequencing libraries were prepared from purified complexes essentially per, Sinha and Winther 2020. Amplified libraries were sequenced by Illumina Miseq.
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| Contributor(s) |
Winther KS, Sinha AK |
| Citation(s) |
33790389 |
| |
| Submission date |
May 12, 2020 |
| Last update date |
Apr 20, 2021 |
| Contact name |
Kristoffer Skovbo Winther |
| E-mail(s) |
kristoffer.winther@bio.ku.dk
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| Organization name |
University og Copenhagen
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| Department |
Department of Biology
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| Street address |
Ole Maaloesvej 5
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| City |
Copenhagen |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platforms (1) |
| GPL16085 |
Illumina MiSeq (Escherichia coli) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA632006 |
| SRA |
SRP261322 |
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