Transcription rates are regulated by the interactions between RNA polymerase, sigma factor, and promoter DNA sequences in bacteria. However, it remains unclear how non-canonical sequence motifs collectively control transcription rates. Here, we combine massively parallel assays, biophysics, and machine learning to develop a 346-parameter model that predicts site-specific transcription initiation rates for any σ70promoter sequence, validated across 22132 bacterial promoters with diverse sequences. We apply the model to predict genetic context effects, design σ70promoters with desired transcription rates, and identify undesired promoters inside engineered genetic systems. The model provides a biophysical basis for understanding gene regulation in natural genetic systems and precise transcriptional control for engineering synthetic genetic systems.
- Award ID(s):
- 1803055
- NSF-PAR ID:
- 10354042
- Editor(s):
- Faust, Karoline
- Date Published:
- Journal Name:
- mSystems
- Volume:
- 6
- Issue:
- 4
- ISSN:
- 2379-5077
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract -
Abstract The Spo0A transcription factor is activated by phosphorylation in starving
Bacillus subtilis cells. The activated Spo0A (Spo0A~P) regulates genes controlling entry into sporulation and appears to control mother‐cell‐specific gene expression after asymmetric division, but the latter remains elusive. Here, we found that Spo0A~P directly binds to three conserved DNA sequences (0A1‐3) in the promoter region of the mother cell‐specific lytic transglycosylase genespoIID , which is transcribed by σE‐RNA polymerase (RNAP) and negatively controlled by the SpoIIID transcription factor and required for forespore engulfment. Systematic mutagenesis of the 0A boxes revealed that the 0A1 and 0A2 boxes located upstream of the promoter positively control the transcription ofspoIID . In contrast, the 0A3 box located downstream of the promoter negatively controls the transcription ofspoIID . The mutated SpoIIID binding site located between the −35 and −10 promoter elements causes increased expression ofspoIID and reduced sporulation. When the mutations of 0A1, 0A2, and IIID sites are combined, sporulation is restored. Collectively, our data suggest that the mother cell‐specificspoIID expression is precisely controlled by the coordination of three factors, Spo0A~P, SpoIIID, and σE‐RNAP, for proper sporulation. The conservation of this mechanism across spore‐forming species was discussed. -
Crosson, Sean (Ed.)Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae , one quorum-sensing receptor and transcription factor, called VqmA (VqmA Vc ), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmA Phage that activates transcription of the phage gene qtip , and Qtip launches the phage lytic program. Curiously, VqmA Phage can activate vqmR expression but VqmA Vc cannot activate expression of qtip . Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmA Phage and A192, the equivalent residue in VqmA Vc , in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmA Phage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmA Phage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmA Phage and VqmA Vc . A second, targeted genetic screen revealed a region located in the VqmA Vc DNA-binding domain that is necessary to prevent VqmA Vc from binding the qtip promoter, thus restricting DNA binding to the vqmR promoter. We propose that the VqmA Vc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmA Vc from binding the qtip promoter.more » « less
-
null (Ed.)Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ luxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a Δ rpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in Δ luxO showed all five qrr genes were repressed, while in Δ rpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species indicating it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ rpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR . Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2 . Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation. Importance The quorum sensing non-coding sRNAs are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in V. harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in V. cholerae the qrr genes are redundant in function. In V. parahaemolyticus , qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR.more » « less
-
Abstract ARGONAUTES are the central effector proteins of
RNA silencing which bind target transcripts in a smallRNA ‐guided manner.Arabidopsis thaliana has 10 (ARGONAUTE ) genes, with specialized roles inAGO RNA ‐directedDNA methylation, post‐transcriptional gene silencing, and antiviral defense. To better understand specialization among genes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters ofAGO ,AGO 1 , andAGO 10 using yeast 1‐hybrid assays. A ranked list of candidateAGO 7DNA ‐bindingTF s revealed binding of the promoter by a number of proteins in two families: the miR156‐regulatedAGO 7SPL family and the miR319‐regulatedTCP family, both of which have roles in developmental timing and leaf morphology. Possible functions forSPL andTCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern of , nor for the function ofAGO 7 in leaf shape. NormalAGO 7 transcription levels and function appear to depend instead on an adjacent 124‐bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conservedAGO 7AGO 7‐triggered pathway functions in timing and polarity.TAS 3