Early work identified two promoter regions, the -10 and -35 elements, that interact sequence specifically with bacterial RNA polymerase (RNAP). However, we now know that several additional promoter elements contact RNAP and influence transcription initiation. Furthermore, our picture of promoter control has evolved beyond one in which regulation results solely from activators and repressors that bind to DNA sequences near the RNAP binding site: many important transcription factors bind directly to RNAP without binding to DNA. These factors can target promoters by affecting specific kinetic steps on the pathway to open complex formation, thereby regulating RNA output from specific promoters.

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We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the ؊10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with 1.2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases at the junction of the ؊10 element and the discriminator region in an unregulated rRNA promoter variant and in the PR promoter. Our studies indicate that nontemplate strand bases adjacent to the ؊10 element bind within a 9-aa interval in 1.2 (residues 99 -107). We also demonstrate that the downstream-most base on the nontemplate strand of the ؊10 hexamer cross-links to region 2, and not to 1.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation.promoter element ͉ RNA polymerase ͉ transcription initiation ͉ discriminator region ͉ Ϫ10 element I nteractions between bacterial RNA polymerase holoenzyme (RNAP; ␣ 2 ␤␤Ј ) and the promoter can determine not only its basal strength but also its regulation. (In this report, always refers to 70 , the major factor.) Four promoter elements are generally recognized as making sequence-specific contacts with RNAP (1, 2) ( Fig. 1): the UP element (bound by the C-terminal domains of the two ␣ subunits); the Ϫ35 hexamer (bound by region 4.2); the extended Ϫ10 element (bound by region 3.0); and the Ϫ10 hexamer (bound by region 2.3-2.4). Recently, an additional element immediately downstream of the Ϫ10 hexamer, within the discriminator region, was proposed to bind to region 1.2 (3, 4).The term ''discriminator'' was coined by Travers (5) more than 25 years ago to describe a GϩC-rich region downstream from the Ϫ10 hexamer in stable RNA (rRNA and tRNA) promoters, and it was proposed that the GϩC content of this region was important for maintaining proper regulation of stable RNA promoters (6, 7). High GϩC content was proposed to impede strand separation, leading to promoter regulation. However, it was found that a C to G substitution 2 nt downstream from the Ϫ10 hexamer in the rRNA promoter rrnB P1 (rrnB P1 C-7G) eliminated its regulation, suggesting that the actual sequence of the discriminator region, in addition to its high GϩC content, is crucial for control of transcription (3).Footprinting, photocross-linking, and genetic approaches led to the conclusion that the nontemplate strand base two positions downstream from the Ϫ10 element in the rrnB P1 C-7G promoter contacts 1.2. When the base at the analogous position in all other promoters investigated was a C (either naturally or by mutation), competitor-resistant complexes formed with RNAP were much shorte...

SummaryIdentification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high resolution x-ray structure defined a ppGpp binding site on T. thermophilus RNAP. We report here effects of ppGpp on ten mutant E. coli RNAPs with substitutions for the analogous residues within 3-4Å of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally-reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of ω, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli rRNA transcription initiation and highlight the importance of inclusion of ω in bacterial RNAP preparations.

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