Gene expression is a highly controlled process that is crucial for normal development. Throughout biology, this process can be regulated through the selection of transcription start sites and the control of transcription initiation. We study a simple system, E. coli RNA polymerase and its interactions with transcription factors, to discern mechanisms that affect transcription initiation. ? ? E. coli RNA polymerase consists of a core (beta, beta?, 2 alphas, and an omega), which has RNA synthesizing activity, and a sigma factor, which recognizes the DNA promoter elements that set the start site for transcription initiation. The primary sigma of E. coli, sigma70, is used during exponential growth and belongs to a large family of prokaryotic primary sigma factors related to each other by sequence, structure, and function. Primary sigma factors have four regions of similarity (regions 1-4). Specific DNA binding can be conferred by residues in region 2, which recognize a -10 element (TATAAT), residues in region 3, which recognize an extended TGn -10 motif (positions -15 to ?13), and residues in region 4, which recognize a -35 element (TTGACA). In addition to the sequence of the elements, the distance between the elements is important for promoter recognition. The -35 and -10 elements are ideally separated by a spacer length of 17 bp. This distance is set in part by the interaction of region 4 with the b-flap structure in core. In addition, the transcriptional start site is typically located 7 nucleotides downstream of the -10 element [-12 TATAAT ?7. Two general categories of E. coli sigma70-dependent promoters have been well-characterized. One group, -10/-35 promoters, has good matches to the canonical -10 and -35 sequences. The other group, extended -10 or TGn/-10 promoters has the TGn motif and an excellent match to the -10 consensus sequence. ? ? We previously identified an unusual promoter, Pminor, present in bacteriophage T4 DNA. Examination of the Pminor promoter sequence failed to locate good matches to any of the typical sigma70 DNA elements at proper positions relative to the transcriptional start site, which had been determined by primer extension. Nonetheless, recognition of Pminor is specific for polymerase containing sigma70, as it is not recognized by polymerase containing the closely related stationary phase sigma factor, sigma38. In addition, Pminor is of interest because the formation of stable polymerase/Pminor complexes increases when sigma70 lacks the N-terminal 99 residues (region 1.1); other tested promoters have been either unaffected or negatively affected by the lack of sigma70 region 1.1. ? ? In the past year, we have defined the minimal Pminor promoter, and showed that it functions in vivo as well as in vitro. We have found that transcription from Pminor incorporates three nontemplated ATPs at the 5? end of the Pminor transcript, which results in an anomalous assignment for the start site when using primer extension analysis. Using the correct assignment of the Pminor +1 start, we observe good matches to the ?35 and TGn elements, but an extremely poor ?10 element. To understand how the elements of Pminor are recognized and used by polymerase containing sigma70, we have investigated how specific mutations within the Pminor promoter region affect transcription. We have found that Pminor mutations in either the -35 or the TGn motif eliminate its activity. Mutation of the TGn motif was compensated by mutations that make the -10 element more canonical, thus converting the -35/TGn promoter to a -35/-10 promoter. Our potassium permanganate footprinting analyses have indicated that when polymerase is in a stable complex with Pminor, the DNA is single-stranded from -6 to +4 and from -11 to +3 on the nontemplate and template strands, respectively. Thus, Pminor represents one of the few -35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better characterized -10/-35 and extended -10 promoters used by E. coli RNA polymerase.? ? ? During infection by T4, E. coli RNA polymerase containing sigma70 is appropriated by the T4 factors MotA and AsiA, which results in the activation of T4 middle promoters. T4 middle promoters contain the sigma70 ?10 DNA element. However, these promoters lack the sigma70 ?35 element, having instead a MotA box centered at ?30, which is bound by MotA. Our previous work has indicated that both AsiA and MotA interact with region 4 of sigma70, the C-terminal portion that normally contacts ?35 DNA as well as the beta-flap structure in core. AsiA binding prevents the sigma70/beta-flap and sigma70/-35 DNA interactions, inhibiting transcription from promoters that require a ?35 element. The N-terminal domain (NTD) of MotA interacts with the far C-terminal region of sigma70. Specifically, we have shown using a 2-hybrid assay that replacing the last 17 residues of sigma70 with the corresponding residues of stationary phase sigma, sigma38, eliminates the interaction of the NTD of MotA with sigma region 4, but decreases the interaction with AsiA by only about 50%. We have now tested this mutant in transcription assays. We found that AsiA inhibition was normal and a 2-fold increase in transcription observed with MotA alone was present, but MotA/AsiA activation was significantly reduced. This result supports the idea that during sigma70 appropriation by MotA and AsiA, the interaction of the far C-terminal portion of sigma70 with MotA is crucial for activation. However, the increase in transcription observed with MotA alone as well as the low level of MotA/AsiA activation that was still seen with the mutant was unexpected. These results suggest that either a very poor interaction of MotA with the C-terminal portion of sigma70 is somewhat productive for transcription or there is another interaction site for MotA, besides the far C-terminal portion, that can promote transcription. ? ? Region 4 of sigma38 differs from region 4 of sigma70 at many residues besides the far C-terminal portion. These differences include those that interact with AsiA in a reported AsiA/sigma region 4 structure. Thus, we also constructed and tested another sigma70/sigma38 chimera in which all of sigma70 region 4 was replaced with region 4 of sigma38. The addition of either AsiA alone or MotA and AsiA together had no significant effect on transcription when using this mutant protein, indicating that sigma38 region 4 is not competent for AsiA inhibition or MotA/AsiA activation. Furthermore, the 2-fold increase in transcription seen with MotA alone was also eliminated when using this mutant.? ? The primary sigma of B. pertussis also varies from sigma70 at many residues, including several in region 4. However, all of the residues that contact AsiA in the sigma70 region 4/AsiA structure are identical in the E. coli and B. pertussis primary sigma proteins. In contrast, the far C-terminal region of the pertussis sigma differs at several residues from that of sigma70. Despite these differences, we found that polymerase containing the pertussis sigma was significantly activated by MotA/AsiA. This result suggests that none of the differences between the far C-terminal portions of the pertussis and E. coli sigmas are crucial for an interaction with MotA. This result provides a good starting point to begin an analysis of the specific sigma 70 residues that contact MotA.
