RNAP transcription cycle and the effect of rifampin. (A) Mechanisms of transcription initiation and rifampin binding. RNAP (blue) binds DNA to form a closed complex with affinities that differ among promoters by at least a factor of 100. The closed complex isomerizes to an open complex at rates that differ among promoters by at least a factor of 100. The open complex is stable at some promoters; at others it collapses back to the closed complex unless it binds initiating nucleoside triphosphate substrates. The competition between abortive initiation and promoter escape (initiation) is highly variable among promoters. Rifampin can bind to all species of RNAP except the elongation complex and traps RNAP in open complexes by forcing the release of 2- to 3-nt abortive transcripts. (B) Effect of rifampin on growth and RNAP location. E. coli cells were cultured, and the absorbance was determined at given time intervals. Rifampin (150 μg/ml) was added at 200 min to one culture (▪), while the other was allowed to continue growing as a control (⧫). After 20 min, the culture with rifampin added stopped growing, while the control continued in exponential growth. The inset shows the results of quantitative PCR using primers designed to amplify in the promoter, 16S, or 23S subunit genes from all seven rRNA operons. Cells were treated with rifampin for 0, 20, or 40 min followed by immunoprecipitation using antibody for RNAP β′ core subunit. The results of PCR performed on genomic DNA before immunoprecipitation are shown on the right end of the gel image, labeled “input.” The quantified relative intensity of each 16S and 23S band is given below the gel image.

Reproducibility of RNA polymerase chIP-chip data. Log₂ ratios of immunoprecipitated versus no-antibody control DNAs from three hybridizations are plotted against each other. A cluster of outliers in biological replicate 1 consists almost entirely of probes from rRNA gene operons. corr. coef., correlation coefficient.

A sample of RNA polymerase chIP-chip data. In both panels, genome position is indicated at the top and ORFs are shown at the bottom, with those above the line being in the forward orientation and those on bottom being the reverse. (A) rRNA-encoding genes showed a different pattern of distribution in biological replicate 1. The smoothed log₂ ratios from the three individual biological replicates are shown, along with the peaks identified in this region. (B) A sample from another region shows the combined log₂ ratios from all biological replicates before and after smoothing with a moving average over 500 bp.

Poor correlation between the magnitude of the RNAP chIP-chip log₂ ratio, gene expression, and promoter similarity to the consensus matrix. (A) The RNAP binding site log₂ ratio was plotted against the similarity of the promoter to consensus. Of 681 known σ70 promoters from RegulonDB, 497 were located within 1,000 bp of an RNAP binding site in chIP-chip data. For 413 of these, a score was available measuring the similarity of the promoter −10 and −35 boxes to consensus, plotted on the vertical axis. The score was calculated by RegulonDB as described previously (21) by establishing a consensus from 584 known promoters and then scoring each promoter by the use of this consensus matrix. (B) For each of the 1,139 RNAP binding sites (peaks) detected by chIP-chip, the log₂ ratio was plotted against gene expression of the nearest downstream ORF. Gene expression data from reference 14 were generated from three biological replicates with Affymetrix arrays using the same growth conditions as for chIP-chip (M9 minimal medium plus glucose at mid-log phase).

Density and periodicity of RNA polymerase binding sites in glucose minimal medium as detected by chIP-chip. (A) The log₂ ratios of 1,139 binding sites were averaged in each 1-kb section of the genome. Those sections lacking a binding site were assigned a value of zero. These data were examined for periodicity by wavelet analysis, which smoothes the data over a range of window sizes to reveal the wavelengths at which the data fluctuates most consistently. The period (wavelength) was plotted against genome position, with colors indicating the magnitude of average log₂ ratio on a scale from white to yellow to red to black. The data were randomized and reanalyzed 1,000 times, allowing the estimation of significance of the original plot. Areas of the wavelet plot where the false-discovery rate was <10% are highlighted, while other areas are faded (5). (B) Profile of 660-kb periodicity. The average log₂ ratios per kilobase were smoothed with a moving average window size of 330 kb. The vertical axis indicates the number of standard deviations by which the data differed from the mean (mean = 0.177; standard deviation = 0.027). Regions marked in red and green indicate positive and negative phases of periodicity at wavelength of 660 kb from panel A above. (C) The same as panel B above, but instead of average log₂ ratios from chIP-chip, the log₂ signal from gene expression measurements in glucose minimal medium was smoothed and plotted (14) (mean = 8.38, standard deviation = 0.32). D) The same as B above, except the number of detected RNAP binding sites per kb was smoothed and plotted (mean = 0.246; standard deviation = 0.028).

Histogram of the distance from 681 known σ⁷⁰ promoters to the nearest RNA polymerase binding site (peak) observed in chIP-chip data.