Real-Time Footprinting of DNA in the First Kinetically Significant Intermediate in Open Complex Formation by Escherichia Coli RNA Polymerase
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Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase Caroline A. Davis*, Craig A. Bingman*†, Robert Landick*§, M. Thomas Record, Jr.*¶ʈ, and Ruth M. Saecker¶ʈ Departments of *Biochemistry, §Bacteriology, and ¶Chemistry and †Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, WI 53706 Edited by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, and approved March 19, 2007 (received for review November 7, 2006) The architecture of cellular RNA polymerases (RNAPs) dictates that where the relatively slow interconversions between I1 and I2 are transcription can begin only after promoter DNA bends into a deep rate-limiting in both the forward and back directions (2, 3). In the channel and the start site nucleotide (؉1) binds in the active site mechanism shown in Eq. 1,I2 and RPo are characterized by their located on the channel floor. Formation of this transcriptionally resistance to a short challenge with a polyanionic competitor competent ‘‘open’’ complex (RPo)byEscherichia coli RNAP at the such as heparin, which acts to sequester any free RNAP present PR promoter is greatly accelerated by DNA upstream of base pair during the challenge. (In contrast, after a 10 to 20 sec challenge ؊47 (with respect to ؉1). Here we report real-time hydroxyl radical with heparin, I1 complexes, which are in rapid equilibrium with (⅐OH) and potassium permanganate (KMnO4) footprints obtained free RNAP and promoter sequences, are eliminated from the under conditions selected for optimal characterization of the first population.) Given the high degree of conservation of bacterial RNAP and promoter DNA sequences, this mechanism is likely kinetically significant intermediate (I1)inRPo formation. ⅐OH foot- .prints reveal that the DNA backbone from ؊71 to ؊81 is engulfed to describe the key steps in initiation in most prokaryotes Moreover, conservation of many elements of sequence, struc- by RNAP in I1 but not in RPo; downstream protection extends to ture, and/or function between bacterial and eukaryotic polymer- -approximately ؉20 in both complexes. KMnO4 footprinting de tects solvent-accessible thymine bases in RP , but not in I .We ase (pol II) subunits and transcription factors supports the o 1 inference that the bacterial intermediates may be homologs of conclude that upstream DNA wraps more extensively on RNAP in initiation intermediates formed by pol II (4, 5). I than in RP and that downstream DNA (؊11 to ؉20) occupies the 1 o Recently we (6) and Ross and Gourse (7) found that the presence active-site channel in I1 but is not yet melted. Mapping of the of DNA upstream of the Ϫ35 promoter recognition hexamer footprinting data onto available x-ray structures provides a de- greatly accelerates (up to Ϸ60-fold) the rate-determining isomer- tailed model of a kinetic intermediate in bacterial transcription ization step (conversion of I1 to I2). Strikingly, DNase I footprinting initiation and suggests how transient contacts with upstream DNA of I1 at the strong bacteriophage promoter PR reveals that when in I1 might rearrange the channel to favor entry of downstream nonspecific DNA upstream of base pair Ϫ47 is present, downstream duplex DNA. DNA is protected to around ϩ20, and thus bound in the active-site BIOCHEMISTRY channel of RNAP. However, when DNA upstream of Ϫ47 is initiation ͉ transcription ͉ wrapping ͉ kinetics deleted, I1 is now less ‘‘advanced,’’ with downstream protection ending at ϩ2 (template strand)/ϩ7 (nontemplate strand) (6). How pecific transcription initiation by Escherichia coli RNA poly- does DNA upstream of Ϫ47 alter downstream interactions in early ␣ Ј ϩ 70 ϭ transcription intermediates? Surprisingly, protection of DNA in I Smerase (RNAP: core subunit composition 2 1 holoenzyme) at promoter sequences is determined by recognition from DNase I cleavage extends upstream only to Ϫ52 (template) on of DNA (Ϫ10 and Ϫ35 hexamers) upstream of the start site (ϩ1) ‘‘full-length’’ PR, similar to what is observed in RPo (6). Because by the specificity subunit 70. Subsequent to binding, a series of DNase I might displace weak upstream interactions in I1 (8, 9), large-scale conformational changes in both RNAP and promoter other techniques are required to probe RNAP–DNA contacts in this transient intermediate. DNA create the initiation-competent open complex (RPo) (1). During these steps, the multisubunit bacterial RNAP acts as an Sclavi et al. (10) recently reported an elegant ‘‘real-time’’ foot- intricate molecular machine and opens Ϸ14 bp of the DNA double printing study of RPo formation at the T7A1 promoter using x-ray-generated ⅐OH and rapid quench mixing. Unlike DNase I, helix. Defining the cascade of conformational changes that occur ⅐OH is small and nonperturbing of weak interactions. It reacts during initiation is essential to understand sequence- and factor- rapidly and exhibits little if any sequence specificity, making it very dependent regulation of the rate of transcription initiation and has useful for probing transient protein–DNA interactions (11). Addi- important applications in chemical biology and in antibiotic design. tionally, the rapid rate of ⅐OH reaction with the DNA backbone However, the intermediates on this pathway are relatively unstable means that fractional protection is approximately proportional to and short-lived and hence are difficult to trap unambiguously. To date, all structural information about complexes known to be on-pathway intermediates in RPo formation has come from chem- Author contributions: C.A.D., M.T.R., and R.M.S. designed research; C.A.D., C.A.B., M.T.R., ical and enzymatic DNA footprinting methods. and R.M.S. performed research; R.L. contributed new reagents/analytic tools; C.A.D., C.A.B., Quantitative kinetic–mechanistic studies find that at least two M.T.R., and R.M.S. analyzed data; and C.A.D., C.A.B., R.L., M.T.R., and R.M.S. wrote the paper. kinetically significant intermediates, generically designated I1 and The authors declare no conflict of interest. I2, precede formation of RPo by E. coli RNAP: This article is a PNAS Direct Submission. Abbreviations: RNAP, RNA polymerase; RPo, open complex; I1, first kinetically significant intermediate; CTD, C-terminal domain; NTD, N-terminal domain. ʈTo whom correspondence may be addressed at: University of Wisconsin, 433 Babcock Drive, Madison, WI 53706. E-mail: [email protected] or [email protected]. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0609888104/DC1. © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609888104 PNAS ͉ May 8, 2007 ͉ vol. 104 ͉ no. 19 ͉ 7833–7838 Downloaded by guest on October 1, 2021 Template strand Nontemplate strand a3500 b 2000 +20 I1 I1 3000 RP RP0 0 Free DNA Free DNA Fig. 1. Hydroxyl radical (⅐OH) foot- 2500 1500 prints of RNAP–promoter complexes. -76 Shown are representative scans of ytisnetnI 2000 -71 ⅐OH cleavage of RNAP–PR promoter -60 -41 -2 -36 1000 -23 mixtures at 17°C at short (25 sec, 1500 Ͼ70% I1, Ͻ30% RPo; red) and long (Ͼ2,500 sec, Ͻ5% I1, Ͼ95% RPo; blue) 1000 -70 -47 -60 500 times after mixing, compared with the cleavage of free DNA (green) on 500 the template strand (a) and nontem- 0 0 plate strand (b). (c) The average pro- Downstream Upstream Upstream Downstream tection in four to five independent c experiments mapped onto the PR se- quence. (See SI Tables 1 and 2 for I1 NT protection at each position observed -80 -70 -60 -50 -40 -30 -20 -10 +1 +10 +20 +30 in each independent experiment.) T Numbers above the DNA cleavage products specifying fragment length in a and b and in between the tem- RP0 plate and nontemplate PR sequences NT -80 -70 -60 -50 -40 -30 -20 -10 +1 +10 +20 +30 in c correspond to promoter position T numbered with respect to the tran- scription start site (ϩ1). (The ⅐OH en- Weak protection hancement observed at ϩ20 on the Moderate protection Strong protection template strand in RPo is not observed Enhancement in any other ⅐OH footprints.) fractional occupancy for a short-lived, rapidly equilibrating inter- Results mediate like I1 (10). By following the appearance of protection of Comparative Analysis of DNA Backbone Interactions in I1 and in RPo the DNA backbone as a function of time (milliseconds to seconds) by ⅐OH Footprinting. How does the presence of upstream DNA after mixing at 37°C, this study (10) circumvented potential issues extend the protection of downstream DNA from ϩ2 (nontem- raised by trapping promoter complexes at low temperature (11) plate)/ϩ7 (template) to approximately ϩ20 in I1 (6)? Are direct and possible displacement of weak upstream interactions by DNase contacts between RNAP and DNA upstream of base pair Ϫ47 I (8, 9). responsible? Fig. 1 summarizes the ⅐OH footprints of I1 and of RPo In the absence of detailed kinetic–mechanistic information for at PR obtained at 25 sec (Ͼ70% I1) and Ͼ2,500 sec (Ͼ95% RPo) this promoter, Sclavi et al. (10) used the footprinting data from base after mixing, respectively (see Methods). Striking differences exist pair Ϫ60 to ϩ20 to infer both the mechanism (e.g., sequence of between these footprints in the upstream boundary, in the degree intermediates and rate constants of their interconversions) and of protection of the far-upstream region of DNA (approximately structure of intermediates preceding RPo. Evidence was obtained Ϫ65 to Ϫ81) and in the degree of protection of the downstream for three classes of intermediates (and two to three subclasses of the DNA (approximately Ϫ17 to ϩ16). Regions of promoter DNA two early intermediates). The mechanistic analysis revealed that, in from Ϫ82 (nontemplate)/Ϫ81 (template) to approximately ϩ20 are general, multiple complexes are populated at T7A1 at each time protected in I1, whereas the upstream boundary of moderate DNA point in the kinetics.