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The Promoter Spacer Influences Transcription Initiation Via 70 Region

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The Promoter Spacer Influences Transcription Initiation Via 70 Region The promoter spacer influences transcription initiation via ␴70 region 1.1 of Escherichia coli RNA polymerase India G. Hook-Barnard and Deborah M. Hinton1 Gene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Sankar Adhya, National Institutes of Health, Bethesda, MD, and approved December 3, 2008 (received for review August 20, 2008) Transcription initiation is a dynamic process in which RNA poly- (11) and major conformational changes (isomerization) of the merase (RNAP) and promoter DNA act as partners, changing in polymerase (Fig. 1A) (12–14). The result of these changes generates response to one another, to produce a polymerase/promoter open a complex in which the promoter is unwound from Ϫ11 to around complex (RPo) competent for transcription. In Escherichia coli ϩ3, and the protection footprint extends to around ϩ25 (9, 11, RNAP, region 1.1, the N-terminal 100 residues of ␴70, is thought to 15–21). In addition, RPo is normally competitor resistant, although occupy the channel that will hold the DNA downstream of the RPo at the very strong ribosomal promoters does not follow this transcription start site; thus, region 1.1 must move from this rule (22, 23). channel as RPo is formed. Previous work has also shown that Much work has been done to understand the conformational region 1.1 can modulate RPo formation depending on the pro- changes that occur as RPc transitions to RPo (reviewed in ref. 1). moter. For some promoters region 1.1 stimulates the formation of In addition, structures of ␴, core polymerase, and holoenzyme from open complexes; at the Pminor promoter, region 1.1 inhibits this thermophilic bacteria (4, 24–29) or portions of E. coli ␴70 (30, 31) formation. We demonstrate here that the AT-rich Pminor spacer have provided 3D scaffolds on which to model these steps. Kinetic sequence, rather than promoter recognition elements or down- analyses using the ␭ promoter PR have revealed transcriptional stream DNA, determines the effect of region 1.1 on promoter intermediates in the pathway from RPc to RPo (refs. 12–14, 20, and ␴70 activity. Using a Pminor derivative that contains good -depen- 32 and references therein). Initially, the ds promoter DNA is dent DNA elements, we find that the presence of a more GC-rich thought to lie across the polymerase, making sequence-specific spacer or a spacer with the complement of the Pminor sequence contacts with ␴70 and the ␣-CTDs. The interaction of the DNA with results in a promoter that is no longer inhibited by region 1.1. the downstream DNA channel (portions of ␤ and ␤Ј) generates an Furthermore, the presence of the P spacer, the GC-rich spacer, minor early intermediate (I1), which, like RPc, is unstable and competitor or the complement spacer results in different mobilities of pro- sensitive. The DNA then moves deeper into the DNA channel moter DNA during gel electrophoresis, suggesting that the spacer through extensive interactions with portions of ␤ and ␤Ј, forming a regions impart differing conformations or curvatures to the DNA. competitor-resistant intermediate, I2. Finally, the DNA around the We speculate that the spacer can influence the trajectory or ϩ1 site begins to melt, and ␴70 region 2.3 contacts single-stranded flexibility of DNA as it enters the RNAP channel and that region 1.1 (ss)DNA bases at positions Ϫ10 through Ϫ7 on the nontemplate acts as a ‘‘gatekeeper’’ to monitor channel entry. strand. For some promoters, contract(s) between residues in ␴70 region 1.2 and ss bases at Ϫ5 and Ϫ6 occurs also (22, 33). The ranscription initiation is a multistep process that requires both protein/ssDNA interactions stabilize the polymerase/promoter Trecognition of promoter DNA and structural isomerization of complex, allowing the template strand to descend into the active site the RNA polymerase (RNAP)/promoter complex to form a ma- of core and the dsDNA downstream to fully enter the downstream chine competent for transcription (reviewed in refs. 1–4). This channel. RPo is achieved when portions of ␤ and ␤Ј, designated the process must be flexible enough to initiate transcription at a variety polymerase ‘‘jaws,’’ close onto the downstream DNA, securing the of promoter sequences yet rigid enough to provide specificity. In DNA within polymerase. bacteria, the ␴ subunit of RNAP holoenzyme is the primary factor ␴70 region 1.1 does not contact DNA, but is thought to play a that sets this specificity. Although bacteria can have multiple ␴ crucial role in the transition from RPc to RPo (34, 35). At some ␴ ␴70 factors, the primary , such as Escherichia coli , is responsible for promoters (␭PR,Ptac,PRNAI) region 1.1 is needed for efficient the expression of housekeeping genes during exponential growth (5, formation of the open complex (34, 35). However, at the Pminor 6). All ␴ factors share related regions 2, 3, and 4, but only primary promoter the rate of RPo formation is actually inhibited by region ␴ proteins have a related, negatively-charged N-terminal portion, 1.1 (34). Although the structure of region 1.1 of Thermophilus region 1.1 (6). maritima has been reported (59), the structure of ␴70 region 1.1 has Transcription initiation begins with the initial binding of RNAP yet to be determined, presumably because its flexibility has made to dsDNA elements to form the polymerase/promoter closed crystallization difficult. However, FRET data modeled with struc- complex (RPc) (7–9) (reviewed in ref. 1) (Fig. 1A). In RPc, tural analyses indicate that in holoenzyme, region 1.1 lies within the polymerase interacts with a fully ds promoter (P). Promoter rec- channel that will be occupied by the downstream DNA when RPo ognition can arise from interactions between the C-terminal do- is formed (Fig. 1A) (36). Consequently, region 1.1 must move for mains (CTDs) of the ␣-subunits (␣-CTDs) and ds promoter se- quences between Ϫ40 and Ϫ60 (UP elements), between ␴70 region 4 and a Ϫ35 element, between ␴70 region 3 and sequences at Ϫ15, Author contributions: I.G.H.-B. and D.M.H. designed research; I.G.H.-B. performed re- Ϫ14 (the extended Ϫ10 motif), and between ␴70 region 2.4 (a search; I.G.H.-B. and D.M.H. analyzed data; and I.G.H.-B. and D.M.H. wrote the paper. portion of region 2) and sequences at Ϫ12/Ϫ11 (the 5Ј end of the The authors declare no conflict of interest. Ϫ10 element) (reviewed in ref. 1). The RPc, which is usually This article is a PNAS Direct Submission. unstable and competitor sensitive, gives an abbreviated protection 1To whom correspondence should be addressed. E-mail: [email protected]. footprint that does not include DNA downstream of the transcrip- This article contains supporting information online at www.pnas.org/cgi/content/full/ tion start site (7, 9, 10). Creation of the stable polymerase/promoter 0808133106/DCSupplemental. BIOCHEMISTRY open complex (Rpo) requires bending and unwinding of the DNA © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808133106 PNAS ͉ January 20, 2009 ͉ vol. 106 ͉ no. 3 ͉ 737–742 Downloaded by guest on September 30, 2021 Fig. 1. Process of transcription initiation, promoter sequences, and transcription with Pmin,Pmin7, and fl ⌬1.1 Pmin11 with E␴ and E␴ (A) Diagram depicting poly- merase promoter contacts in RPc and RPo with core 70 polymerase (␤, ␤Ј, ␣2, and ␻) in purple, ␴ regions 2–4 in white, ␴70 region 1.1 in green, and DNA in red. R is RNA polymerase; P is the promoter DNA. The transcrip- tional start site is designated ϩ1. Interactions between the ␣-CTDs and the UP element(s), ␴70 region 4 and the Ϫ35 element, ␴70 region 3 and the Ϫ15TGnϪ13 element, and ␴70 region 2 and the Ϫ10 element are indicated. In RPc, the dsDNA has not yet entered the primary chan- nel; full entry of DNA into the channel is blocked by ␴70 region 1.1. In RPo, ␴70 region 1.1 has moved, the DNA is bent and is unwound from Ϫ11 to ϩ3, the template strand has descended into the active site of polymer- ase, and a portion of ␤,␤Ј, called jaws, has secured the downstream DNA. (B) Sequences of Pmin,Pmin deriva- 70 tives, and PlacUV5-Mut. Consensus sequences for the ␴ - dependent Ϫ35, TGn, and Ϫ10 promoter elements are shown at the top. The EcoRI and SalI restriction sites used for plasmid constructions are boxed. ␴70 elements are shaded in gray, and base-pair substitutions in the Pmin derivatives are in red. (C) Effect of promoter mu- tations on activity with E␴fl or E␴⌬1.1. Single-round transcription reactions were performed as described in Materials and Methods using E␴fl (blue) or E␴⌬1.1 (green). The amount of RNA from the indicated pro- moter (relative to the amount of RNA obtained at the 10-min time point with E␴fl ϫ 100) is plotted versus the length of the incubation of polymerase with the DNA (in min) before the addition of rNTPs and heparin. the DNA to occupy the channel as it does in RPo. Kinetic data with of ␴70 region 1.1 varies depending on the specific promoter tested ␭PR suggests that a conformational rearrangement, occurring at the (34). For PuvsX-sigma, region 1.1 has little effect on the rate of open I1 7 I2 transition, is consistent with the movement of 1.1 out of the complex formation. For another promoter, Pminor, region 1.1 sig- channel (12), and FRET analyses have suggested that region 1.1 nificantly inhibits formation of RPo.
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