The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the ␤ subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. RNA polymerase (RNAP) in Escherichia coli is a key factor in gene expression and catalyzes the transcription of DNA to mRNA for all genes (reviewed in references 33 and 37). The core enzyme is composed of four subunits: two ␣, one ␤, one ␤Ј, and one . Core RNAP becomes transcriptionally active holoenzyme with the addition of a factor (Fig. 1A). E. coli possesses seven interchangeable factors, each with specificity for different promoters. Sigma factors function as global regulators of gene expression and mediate the transcriptional response to conditions under which a large number of genes need to be turned on or off, such as stationary phase or heat shock. Except for 54 , sigma factors do not bind to DNA except as part of the holoenzyme complex (9).To map promoters in bacteria, we sought a way to force RNAP to reside only at promoters so that identifying DNA fragments bound to RNAP in vivo would report promoter locations. A variety of small-molecule inhibitors of RNA polymerase were evaluated for the immobilization of RNAP, and rifampin was found to work best (M. Raffaelle, E. Kanin, J. Vogt, R. R. Burgess, A. Z. Ansari, unpublished data). The antibiotic rifampin inhibits bacterial growth by binding the ␤ subunit of RNAP just upstream of the active site, blocking the synthesis of RNAs longer than 2 to 3 nucleotides (nt) (11) (Fig. 1A). Rifampin has no effect on RNAP promoter binding to form closed complexes or on open complex formation (30) and has no effect on RNAP in vitro when added after elongating RNAP has cleared the promoter (12, 38). Upon addition of rifampin to a growing culture, the rifampin stops growth without killing cells by diffusion across bacterial membranes and tight binding to RNAP not engaged in transcription. RNAP holoenzyme complexed with rifampin can still bind to promoters but is trapped in an abortive cycle and unable to extend RNAs beyond 2 to 3 nt (Fig. 1A). However, RNAP molecules in elongation complexes with RNA and DNA are resistant to rifampin binding. Once elongating RNAPs terminate transcription and release RNA and DNA, they become susceptible to rifampin binding, which traps them in newly formed open complexes.A promoter is a DNA sequence to which RNAP binds and initiates the transcription of RNA. Knowledge of promoter locations is the first step in the elucidation of the transcriptional regulatory network. It allows the identification of which genes are cotranscribed and, from the DNA sequence, identification of regulatory motifs associated with different regulators. It also provides a basis for the interpretation of the bindin...

We investigated the binding of E. coli RNA polymerase holoenzymes bearing sigma70, sigma(S), sigma32, or sigma54 to the ribosomal RNA operons (rrn) in vivo. At the rrn promoter, we observed "holoenzyme switching" from Esigma70 to Esigma(S) or Esigma32 in response to environmental cues. We also examined if sigma factors are retained by core polymerase during transcript elongation. At the rrn operons, sigma70 translocates briefly with the elongating polymerase and is released stochastically from the core polymerase with an estimated half-life of approximately 4-7 s. Similarly, at gadA and htpG, operons that are targeted by Esigma(S) and Esigma32, respectively, we find that sigma(S) and sigma32 also dissociate stochastically, albeit more rapidly than sigma70, from the elongating core polymerase. Up to approximately 70% of Esigma70 (the major vegetative holoenzyme) in rapidly growing cells is engaged in transcribing the rrn operons. Thus, our results suggest that at least approximately 70% of cellular holoenzymes release sigma70 during transcript elongation. Release of sigma factors during each round of transcription provides a simple mechanism for rapidly reprogramming polymerase with the relevant sigma factor and is consistent with the occurrence of a "sigma cycle" in vivo.

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a ''switch''-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.DNA recognition ͉ extradenticle ͉ HOX proteins ͉ polyamide