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A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq.

Druzhinin SY, Tran NT, Skalenko KS, Goldman SR, Knoblauch JG, Dove SL, Nickels BE - PLoS Genet. (2015)

Bottom Line: Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation.In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation).Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, New Jersey, United States of America.

ABSTRACT
Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, "primer-dependent initiation," (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5' ends (5' RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.

No MeSH data available.


Related in: MedlinePlus

Detection of PDI in E. coli.A. Analysis of PDI by 5′ RNA-seq. Percentage of transcripts emanating from position −1 of uTSRs with the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wild-type) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). The Nrn effect represents the difference in these values. uTSRs with above average Nrn effect are highlighted in black. The total number of uTSRs used to calculate the percentages is indicated. Values are calculated from biological replicates listed in S8 Table. Data is derived from the analysis of all 5′ ends during stationary phase. B. Analysis of PDI at the promoter associated with tomB, ptomB, by 5′ RNA-seq. Sequence of ptomB is shown. Indicated are positions +1, −1 and the promoter −10 and −35 elements. Graph on the left shows average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs or cells in which the oligoRNase NrnB was ectopically expressed (Nrn) as detected by 5′ RNA-seq analysis of all 5′ ends during stationary phase. Graph on the right shows the average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs as detected by 5′ RNA-seq analysis of hydroxyl 5′ ends (OH), monophosphate 5′ ends (P), or triphosphate 5′ ends (PPP) during stationary phase. C. Primer extension analysis of plasmid-borne ptomB variants carrying the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wt) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). D. Primer extension analysis of the plasmid-borne ptomB variant carrying the sequence G−1G+1 during exponential phase (exp) or stationary phase (sta).
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pgen.1005348.g004: Detection of PDI in E. coli.A. Analysis of PDI by 5′ RNA-seq. Percentage of transcripts emanating from position −1 of uTSRs with the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wild-type) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). The Nrn effect represents the difference in these values. uTSRs with above average Nrn effect are highlighted in black. The total number of uTSRs used to calculate the percentages is indicated. Values are calculated from biological replicates listed in S8 Table. Data is derived from the analysis of all 5′ ends during stationary phase. B. Analysis of PDI at the promoter associated with tomB, ptomB, by 5′ RNA-seq. Sequence of ptomB is shown. Indicated are positions +1, −1 and the promoter −10 and −35 elements. Graph on the left shows average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs or cells in which the oligoRNase NrnB was ectopically expressed (Nrn) as detected by 5′ RNA-seq analysis of all 5′ ends during stationary phase. Graph on the right shows the average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs as detected by 5′ RNA-seq analysis of hydroxyl 5′ ends (OH), monophosphate 5′ ends (P), or triphosphate 5′ ends (PPP) during stationary phase. C. Primer extension analysis of plasmid-borne ptomB variants carrying the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wt) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). D. Primer extension analysis of the plasmid-borne ptomB variant carrying the sequence G−1G+1 during exponential phase (exp) or stationary phase (sta).

Mentions: To facilitate a direct comparison of the results obtained in E. coli with those obtained in V. cholerae (Fig 3A), we identified 401 uTSRs (S8 Table). On average, in cells carrying wild-type concentrations of 2- to ~4-nt RNAs, ~11% of the transcripts associated with these 401 uTSRs emanated from position −1 in the analysis of all 5′ ends (Fig 4A). In contrast, the proportion of transcripts initiating from position −1 was reduced to ~4% in cells in which NrnB was ectopically expressed (Fig 4A). Thus, on average, ~7% of the total transcripts associated with a given uTSR are NrnB-sensitive in E. coli. Analysis of the 393 uTSRs where position +1 was an A or G revealed that, as in V. cholerae, NrnB-sensitive transcripts initiating from position −1 are preferentially generated from uTSRs carrying T−1A+1 (an average Nrn-effect of ~20% of the total transcripts for T−1A+1 start site regions versus an average Nrn-effect of ~7% for all start sites regions) and G−1G+1 (an average Nrn-effect of ~18% for G−1G+1 start site regions) (Fig 4A).


A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq.

Druzhinin SY, Tran NT, Skalenko KS, Goldman SR, Knoblauch JG, Dove SL, Nickels BE - PLoS Genet. (2015)

Detection of PDI in E. coli.A. Analysis of PDI by 5′ RNA-seq. Percentage of transcripts emanating from position −1 of uTSRs with the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wild-type) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). The Nrn effect represents the difference in these values. uTSRs with above average Nrn effect are highlighted in black. The total number of uTSRs used to calculate the percentages is indicated. Values are calculated from biological replicates listed in S8 Table. Data is derived from the analysis of all 5′ ends during stationary phase. B. Analysis of PDI at the promoter associated with tomB, ptomB, by 5′ RNA-seq. Sequence of ptomB is shown. Indicated are positions +1, −1 and the promoter −10 and −35 elements. Graph on the left shows average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs or cells in which the oligoRNase NrnB was ectopically expressed (Nrn) as detected by 5′ RNA-seq analysis of all 5′ ends during stationary phase. Graph on the right shows the average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs as detected by 5′ RNA-seq analysis of hydroxyl 5′ ends (OH), monophosphate 5′ ends (P), or triphosphate 5′ ends (PPP) during stationary phase. C. Primer extension analysis of plasmid-borne ptomB variants carrying the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wt) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). D. Primer extension analysis of the plasmid-borne ptomB variant carrying the sequence G−1G+1 during exponential phase (exp) or stationary phase (sta).
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pgen.1005348.g004: Detection of PDI in E. coli.A. Analysis of PDI by 5′ RNA-seq. Percentage of transcripts emanating from position −1 of uTSRs with the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wild-type) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). The Nrn effect represents the difference in these values. uTSRs with above average Nrn effect are highlighted in black. The total number of uTSRs used to calculate the percentages is indicated. Values are calculated from biological replicates listed in S8 Table. Data is derived from the analysis of all 5′ ends during stationary phase. B. Analysis of PDI at the promoter associated with tomB, ptomB, by 5′ RNA-seq. Sequence of ptomB is shown. Indicated are positions +1, −1 and the promoter −10 and −35 elements. Graph on the left shows average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs or cells in which the oligoRNase NrnB was ectopically expressed (Nrn) as detected by 5′ RNA-seq analysis of all 5′ ends during stationary phase. Graph on the right shows the average distribution of 5′ ends between positions −3 and +4 for ptomB in cells carrying wild-type concentrations of 2- to ~4-nt RNAs as detected by 5′ RNA-seq analysis of hydroxyl 5′ ends (OH), monophosphate 5′ ends (P), or triphosphate 5′ ends (PPP) during stationary phase. C. Primer extension analysis of plasmid-borne ptomB variants carrying the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wt) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). D. Primer extension analysis of the plasmid-borne ptomB variant carrying the sequence G−1G+1 during exponential phase (exp) or stationary phase (sta).
Mentions: To facilitate a direct comparison of the results obtained in E. coli with those obtained in V. cholerae (Fig 3A), we identified 401 uTSRs (S8 Table). On average, in cells carrying wild-type concentrations of 2- to ~4-nt RNAs, ~11% of the transcripts associated with these 401 uTSRs emanated from position −1 in the analysis of all 5′ ends (Fig 4A). In contrast, the proportion of transcripts initiating from position −1 was reduced to ~4% in cells in which NrnB was ectopically expressed (Fig 4A). Thus, on average, ~7% of the total transcripts associated with a given uTSR are NrnB-sensitive in E. coli. Analysis of the 393 uTSRs where position +1 was an A or G revealed that, as in V. cholerae, NrnB-sensitive transcripts initiating from position −1 are preferentially generated from uTSRs carrying T−1A+1 (an average Nrn-effect of ~20% of the total transcripts for T−1A+1 start site regions versus an average Nrn-effect of ~7% for all start sites regions) and G−1G+1 (an average Nrn-effect of ~18% for G−1G+1 start site regions) (Fig 4A).

Bottom Line: Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation.In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation).Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, New Jersey, United States of America.

ABSTRACT
Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, "primer-dependent initiation," (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5' ends (5' RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.

No MeSH data available.


Related in: MedlinePlus