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Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

Bonocora RP, Smith C, Lapierre P, Wade JT - PLoS Genet. (2015)

Bottom Line: Strikingly, the majority of σ54 binding sites are located inside genes.We conclude that many intragenic σ54 binding sites are likely to be functional.Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

ABSTRACT
Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

No MeSH data available.


Related in: MedlinePlus

Genome-wide σ54-dependent changes in gene expression.Relative RNA levels, determined by RNA-seq, for all genes in cells transiently overexpressing rpoN (MG1655 ΔrpoN + pRpoN; RPB149) or control cells containing empty vector (MG1655 ΔrpoN + pBAD24; RPB152). Relative RNA levels were calculated using Rockhopper [65]. Each gene is indicated by a grey data point. Genes immediately downstream of intergenic σ54 sites (blue), or genes containing intragenic σ54 sites (red) are highlighted.
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pgen.1005552.g005: Genome-wide σ54-dependent changes in gene expression.Relative RNA levels, determined by RNA-seq, for all genes in cells transiently overexpressing rpoN (MG1655 ΔrpoN + pRpoN; RPB149) or control cells containing empty vector (MG1655 ΔrpoN + pBAD24; RPB152). Relative RNA levels were calculated using Rockhopper [65]. Each gene is indicated by a grey data point. Genes immediately downstream of intergenic σ54 sites (blue), or genes containing intragenic σ54 sites (red) are highlighted.

Mentions: σ54 can bind promoter DNA in the absence of core RNAP in vitro, although the affinity is lower than that in the context of RNAP holoenzyme [5]. To determine whether σ54 binding in vivo is in the context of RNAP holoenzyme, we used ChIP-seq to map the genome-wide distribution of the β subunit of core RNAP under the same growth conditions used for σ54 ChIP-seq. As shown in Fig 1A, we detected increased local signal for β at sites of σ54 binding. In some cases, especially for intragenic σ54 binding sites, the σ54 peak is located within a transcribed region. Hence, it is more difficult to ascertain σ54-dependent RNAP binding at these positions. Therefore, we determined the median occupancy of RNAP at each of the positions in the 1 kbp region surrounding all σ54 binding sites (Fig 4; note that each σ54 peak contributes equally in this analysis, regardless of the ChIP-seq signal for σ54). RNAP binding is substantially greater at the exact site of σ54 binding than in the flanking sequence. Furthermore, RNAP binding at the motif center is enriched compared to RNAP binding at randomly selected positions throughout the genome (Fig 4). We conclude that most or all σ54 binding in vivo is in the context of RNAP holoenzyme. We also noted that the distribution of RNAP binding at σ54 binding sites is symmetric (Figs 1A and 4), indicative of RNAP that is not actively transcribing RNA. Thus, our data strongly suggest that most or all RNAP:σ54 is transcriptionally inactive under the conditions tested. Furthermore, as described in more detail below, little RNA initiates from identified σ54 binding sites under the conditions used in our study (Fig 5 and S4 Table).


Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

Bonocora RP, Smith C, Lapierre P, Wade JT - PLoS Genet. (2015)

Genome-wide σ54-dependent changes in gene expression.Relative RNA levels, determined by RNA-seq, for all genes in cells transiently overexpressing rpoN (MG1655 ΔrpoN + pRpoN; RPB149) or control cells containing empty vector (MG1655 ΔrpoN + pBAD24; RPB152). Relative RNA levels were calculated using Rockhopper [65]. Each gene is indicated by a grey data point. Genes immediately downstream of intergenic σ54 sites (blue), or genes containing intragenic σ54 sites (red) are highlighted.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591121&req=5

pgen.1005552.g005: Genome-wide σ54-dependent changes in gene expression.Relative RNA levels, determined by RNA-seq, for all genes in cells transiently overexpressing rpoN (MG1655 ΔrpoN + pRpoN; RPB149) or control cells containing empty vector (MG1655 ΔrpoN + pBAD24; RPB152). Relative RNA levels were calculated using Rockhopper [65]. Each gene is indicated by a grey data point. Genes immediately downstream of intergenic σ54 sites (blue), or genes containing intragenic σ54 sites (red) are highlighted.
Mentions: σ54 can bind promoter DNA in the absence of core RNAP in vitro, although the affinity is lower than that in the context of RNAP holoenzyme [5]. To determine whether σ54 binding in vivo is in the context of RNAP holoenzyme, we used ChIP-seq to map the genome-wide distribution of the β subunit of core RNAP under the same growth conditions used for σ54 ChIP-seq. As shown in Fig 1A, we detected increased local signal for β at sites of σ54 binding. In some cases, especially for intragenic σ54 binding sites, the σ54 peak is located within a transcribed region. Hence, it is more difficult to ascertain σ54-dependent RNAP binding at these positions. Therefore, we determined the median occupancy of RNAP at each of the positions in the 1 kbp region surrounding all σ54 binding sites (Fig 4; note that each σ54 peak contributes equally in this analysis, regardless of the ChIP-seq signal for σ54). RNAP binding is substantially greater at the exact site of σ54 binding than in the flanking sequence. Furthermore, RNAP binding at the motif center is enriched compared to RNAP binding at randomly selected positions throughout the genome (Fig 4). We conclude that most or all σ54 binding in vivo is in the context of RNAP holoenzyme. We also noted that the distribution of RNAP binding at σ54 binding sites is symmetric (Figs 1A and 4), indicative of RNAP that is not actively transcribing RNA. Thus, our data strongly suggest that most or all RNAP:σ54 is transcriptionally inactive under the conditions tested. Furthermore, as described in more detail below, little RNA initiates from identified σ54 binding sites under the conditions used in our study (Fig 5 and S4 Table).

Bottom Line: Strikingly, the majority of σ54 binding sites are located inside genes.We conclude that many intragenic σ54 binding sites are likely to be functional.Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

ABSTRACT
Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

No MeSH data available.


Related in: MedlinePlus