Limits...
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

Examples of novel σ54 promoters associated with transcription under conditions of nitrogen limitation.Microarray data from [36] showing RNA levels at selected regions around novel (A) intergenic or (B) intragenic σ54 promoters. The σ54 promoter upstream of nac has been previously described [71] and serves as a positive control. Grey arrows represent genes. Grey arrows with dotted lines indicate that only a portion of the gene is shown. Bent, black arrows represent σ54 promoter motifs. Grey and green histograms indicate RNA levels in nitrogen-rich and nitrogen-limiting media, respectively and have been scaled equivalently. Blue histogram indicates σ54 ChIP-seq occupancy. Percentages indicate relative scale on the y-axis.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591121&req=5

pgen.1005552.g006: Examples of novel σ54 promoters associated with transcription under conditions of nitrogen limitation.Microarray data from [36] showing RNA levels at selected regions around novel (A) intergenic or (B) intragenic σ54 promoters. The σ54 promoter upstream of nac has been previously described [71] and serves as a positive control. Grey arrows represent genes. Grey arrows with dotted lines indicate that only a portion of the gene is shown. Bent, black arrows represent σ54 promoter motifs. Grey and green histograms indicate RNA levels in nitrogen-rich and nitrogen-limiting media, respectively and have been scaled equivalently. Blue histogram indicates σ54 ChIP-seq occupancy. Percentages indicate relative scale on the y-axis.

Mentions: Our RNA-seq analysis indicates that only three bEBP activators, NtrC, PspF and GlrR, are active under the conditions used, most likely at a very low level (Fig 5 and S4 Table). A single condition where most bEBPs are induced has not been determined; hence, most σ54 promoters will be inactive under a given condition. The bEBP NtrC is highly active in nitrogen-limiting conditions [20]. We compared our ChIP-seq data to published microarray data from E. coli grown under nitrogen-rich conditions (NtrC inactive), and conditions known to induce activity of NtrC [36]. Not all of the σ54 initiated genes are expected to be up-regulated under these conditions, just those under control of NtrC. There are 15 previously described NtrC-regulated operons in E. coli [20,37]. All of these operons are associated with a σ54 ChIP-seq peak (our study), and most are associated with observed increases in expression in nitrogen-limiting conditions, e.g. nac (Fig 6A) [29]. We observed two additional intergenic ChIP-seq peaks associated with increased expression of the downstream genes, hypA and zraP, in nitrogen-limiting conditions (Fig 6A). hypA and zraP are both known to be transcribed by RNAP:σ54; however, transcription of these genes is activated by bEBPs other than NtrC. Specifically, transcription of hypA is activated by FhlA in response to anaerobiosis and the presence of formate [38], and transcription of zraP is activated by ZraR in response to high zinc concentrations [39]. Thus, our data suggest either that hypA and zraP are regulated by multiple bEBPs (i.e. NtrC and at least one other), or that bEBPs other than NtrC are activated under the growth conditions tested. The latter is more likely since no NtrC binding was detected upstream of hypA or zraP by ChIP-seq [20]. We also observed three transcripts induced under low nitrogen conditions that initiate from intragenic σ54 binding sites. These σ54 binding sites, within nagB, yqeC and rlmD, are located immediately upstream of the start sites for the transcripts induced by nitrogen limitation, and the predicted -24 and -12 motifs are oriented in the same direction as the associated RNAs (Fig 6B). In each case, the RNA that appears to be transcribed by RNAP:σ54 extends through the adjacent gene (Fig 6B). Thus, these RNAs appear to be mRNAs with unusually long 5ʹ UTRs. A recent study identified an NtrC-activated σ54 promoter inside rlmD [20]. This promoter is an exact match to the one we identified, and drives transcription of the entire relA gene (adjacent to rlmD), consistent with our analysis.


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

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

Examples of novel σ54 promoters associated with transcription under conditions of nitrogen limitation.Microarray data from [36] showing RNA levels at selected regions around novel (A) intergenic or (B) intragenic σ54 promoters. The σ54 promoter upstream of nac has been previously described [71] and serves as a positive control. Grey arrows represent genes. Grey arrows with dotted lines indicate that only a portion of the gene is shown. Bent, black arrows represent σ54 promoter motifs. Grey and green histograms indicate RNA levels in nitrogen-rich and nitrogen-limiting media, respectively and have been scaled equivalently. Blue histogram indicates σ54 ChIP-seq occupancy. Percentages indicate relative scale on the y-axis.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005552.g006: Examples of novel σ54 promoters associated with transcription under conditions of nitrogen limitation.Microarray data from [36] showing RNA levels at selected regions around novel (A) intergenic or (B) intragenic σ54 promoters. The σ54 promoter upstream of nac has been previously described [71] and serves as a positive control. Grey arrows represent genes. Grey arrows with dotted lines indicate that only a portion of the gene is shown. Bent, black arrows represent σ54 promoter motifs. Grey and green histograms indicate RNA levels in nitrogen-rich and nitrogen-limiting media, respectively and have been scaled equivalently. Blue histogram indicates σ54 ChIP-seq occupancy. Percentages indicate relative scale on the y-axis.
Mentions: Our RNA-seq analysis indicates that only three bEBP activators, NtrC, PspF and GlrR, are active under the conditions used, most likely at a very low level (Fig 5 and S4 Table). A single condition where most bEBPs are induced has not been determined; hence, most σ54 promoters will be inactive under a given condition. The bEBP NtrC is highly active in nitrogen-limiting conditions [20]. We compared our ChIP-seq data to published microarray data from E. coli grown under nitrogen-rich conditions (NtrC inactive), and conditions known to induce activity of NtrC [36]. Not all of the σ54 initiated genes are expected to be up-regulated under these conditions, just those under control of NtrC. There are 15 previously described NtrC-regulated operons in E. coli [20,37]. All of these operons are associated with a σ54 ChIP-seq peak (our study), and most are associated with observed increases in expression in nitrogen-limiting conditions, e.g. nac (Fig 6A) [29]. We observed two additional intergenic ChIP-seq peaks associated with increased expression of the downstream genes, hypA and zraP, in nitrogen-limiting conditions (Fig 6A). hypA and zraP are both known to be transcribed by RNAP:σ54; however, transcription of these genes is activated by bEBPs other than NtrC. Specifically, transcription of hypA is activated by FhlA in response to anaerobiosis and the presence of formate [38], and transcription of zraP is activated by ZraR in response to high zinc concentrations [39]. Thus, our data suggest either that hypA and zraP are regulated by multiple bEBPs (i.e. NtrC and at least one other), or that bEBPs other than NtrC are activated under the growth conditions tested. The latter is more likely since no NtrC binding was detected upstream of hypA or zraP by ChIP-seq [20]. We also observed three transcripts induced under low nitrogen conditions that initiate from intragenic σ54 binding sites. These σ54 binding sites, within nagB, yqeC and rlmD, are located immediately upstream of the start sites for the transcripts induced by nitrogen limitation, and the predicted -24 and -12 motifs are oriented in the same direction as the associated RNAs (Fig 6B). In each case, the RNA that appears to be transcribed by RNAP:σ54 extends through the adjacent gene (Fig 6B). Thus, these RNAs appear to be mRNAs with unusually long 5ʹ UTRs. A recent study identified an NtrC-activated σ54 promoter inside rlmD [20]. This promoter is an exact match to the one we identified, and drives transcription of the entire relA gene (adjacent to rlmD), consistent with our analysis.

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