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Control of Transcription Initiation


Transcription initiation is a multi-step process relying not only on recognition of promoter DNA, but also requiring structural isomerization of the entire RNA polymerase/promoter complex to form a machine competent for transcription. Much of this process is dependent upon the sigma subunit of the RNA polymerase.<P> In E. coli the primary sigma is sigma70. Sigma70 regions 2.4, 3 and 4.2 contribute to double strand DNA recognition and binding via interactions with the -10, TGn, and -35 promoter elements, respectively. This initial binding of promoter DNA elements by RNA polymerase is termed the closed complex (RPc). For transcription to begin, the RPc must isomerize into the open complex (RPo), in which the DNA is single stranded and the template strand lies in the RNA polymerase active site. Sigma70 regions 2.3, 1.2, and 1.1 play crucial roles in the transient intermediate steps between RPc and RPo. Region 2.3 binds to single-stranded DNA in the -10 element, and region 1.2 contacts nucleotides at position -5. However, region 1.1, the N-terminal 100 amino acids of sigma70, does not contact DNA. Rather, it appears to monitor isomerization of RNA polymerase.<P> Biophysical and biochemical analyses from other labs have indicated that the negatively charged region 1.1 lies within the RNAP channel during RPc. However, in RPo region 1.1 has been displaced by downstream DNA and is located outside the channel. At one promoter that has been studied, region 1.1 is essential for transition to RPo, and the movement of region 1.1 may be coupled to late folding of the polymerase jaws, facilitating isomerization of RNA polymerase into a stable (competitor resistant) RPo. Additionally, movement of region 1.1 could influence contact between region 1.2 and the position -5 nucleotide. <P> Work in our lab has identified an unusual promoter, Pminor, whose initiation of transcription increases when sigma70 lacks region 1.1. Other tested promoters have been either unaffected or negatively affected by the lack of region 1.1. Our mutational analysis of Pminor has indicated that both the -35 element and TGn motif are required for efficient transcription and that these elements compensate for the poor -10 element. Thus, Pminor represents one of only a few characterized -35/TGn promoters.<P> Because the absence of region 1.1 enhances transcription from Pminor, we have used this promoter to investigate the role of region 1.1 using RNA polymerase reconstituted with either full-length sigma or sigma lacking region 1.1. We find that the effect region 1.1 has on promoter activity is not determined by core promoter elements (the -35, TGn, -10 elements). Instead, we observe that the influence of region 1.1 can be altered by changing the AT-richness of a promoters spacer sequence. We also demonstrate, by DNaseI footprinting, that jaw closure is not reliant upon movement of region 1.1, as polymerase lacking region 1.1 is able to protect Pminor DNA to +27 in the presence of competitor, consistent with jaw closure. <P> Our results suggest a crucial role for a promoters spacer region in the isomerization of RNA polymerase to the transcriptionally competent open complex. We speculate that the AT-richness of the spacer could affect isomerization by influencing how easily the DNA bends into the active site channel of polymerase.

Hinton, Deborah
DHHS/NIH - National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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