Though opening of the start site (þ1) region of promoter DNA is required for transcription by RNA polymerase (RNAP), surprisinglylittle is known about how and when this occurs in the mechanism. Early events at the λP R promoter load this region of duplex DNA into the active site cleft of Escherichia coli RNAP, forming the closed, permanganate-unreactive intermediate I 1 . Conversion to the subsequent intermediate I 2 overcomes a large enthalpic barrier. Is I 2 open? Here we create a burst of I 2 by rapidly destabilizing open complexes (RP o ) with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from −11 to þ2 in I 2 are permanganate-reactive, demonstrating that RNAP opens the entire initiation bubble in the cleft in a single step. Rates of decay of all observed thymine reactivities are the same as the I 2 to I 1 conversion rate determined by filter binding. In I 2 , permanganatereactivity oftheþ1 thymineon thetemplate(t) strand is the same as the RP o control, whereas nontemplate (nt) thymines are significantly less reactive than in RP o . We propose that: (i) the þ1ðtÞ thymine is in the active site in I 2 ; (ii) conversion of I 2 to RP o repositions the nt strand in the cleft; and (iii) movements of the nt strand are coupled to the assembly and DNA binding of the downstream clamp and jaw that occurs after DNA opening and stabilizes RP o . We hypothesize that unstable open intermediates at the λP R promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters.bottleneck step | transcription regulation | burst experiment | protein nucleic acid interactions I nteractions between RNA polymerase and specific promoter DNA sequences trigger a precise progression of conformational changes in both biomolecules. Taken together, these steps constitute the mechanism of DNA opening and the start of the transcription cycle. For Escherichia coli RNA polymerase holoenzyme (RNAP, subunit composition: α 2 ββ 0 ωσ 70 ), binding free energy drives opening of the initiation bubble (−11 to þ2, numbering relative to the start site base þ1) in promoter DNA, placement of þ1 template base in the active site of the enzyme, and subsequent conformational changes to form the stable open complex RP o . Each of these steps provides a checkpoint for regulatory input.Over the past decade, structural (X-ray, FRET), singlemolecule, and rapid mixing kinetic studies have greatly advanced the understanding of this machinery and these steps. Key advances include: (i) elucidation of the RNAP architecture at atomic resolution (1-4); (ii) dissection of composite forward and backward rate constants for RP o formation into individual rate and/or equilibrium constants for the steps leading to RP o (5, 6); (iii) single-molecule measurements of DNA topological changes (7) Evidence for at least two kinetically significant intermediates (generically designated I 1 and I 2 ) preceding RP o exists for a variety of promoters recognized by E. coli RNAP (cf. refs. 6,8,[14][15][16][17]. Conversion of I 1 to...

Transcription by all RNA polymerases (RNAP) requires a series of large-scale conformational changes to form the transcriptionally-competent open complex RP o . At the λP R promoter, E. coli σ 70 RNAP first forms a wrapped, closed 100 bp complex I 1 . The subsequent step opens the entire 13 base DNA bubble, creating the relatively unstable (open) complex I 2 . Additional conformational changes convert I 2 to the stable RP o . Here we probe these events by dissecting the effects of Na + salts of Glu − , F − and Cl − on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I 1 at 25 °C is ~30-fold larger in Glu − than in Cl − at the same [Na + ], with the same log-log [salt] dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I 1 to I 2 ) are only weakly [salt]-dependent, and the opening rate constant is insensitive to replacement of Cl − by Glu − . These very small effects of [salt] on a process (DNA opening) which is strongly [salt]-dependent in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process, and/or that compensation is present between ion uptake and release.Replacement of Cl − by Glu − or F − at 25 °C greatly increases the lifetime of RP o and greatly reduces its [salt]-dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu − and F − drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I 2 to RP o , while Cl − does not. Because the Hofmeister effect of these anions largely compensates for the destabilizing coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RP o remains long-lived even at 0.5 M Na + in Glu − or F − salts. The observation that Eσ 70 RP o are exceedingly long-lived in moderate to high [Glu − ] argues that Eσ 70 RNAP do not dissociate from strong promoters in vivo when the cytoplamsic increases during osmotic stress. † This work was supported by NIH grant GM23467 to M.T. R. W. S. K. gratefully acknowledges the support of the Biotechnology Training Program (NIH 5 T32 GM08349); T. G. is the recipient of the William R. and Dorothy E. Sullivan Distinguished Graduate Fellowship. * To whom correspondence should be addressed: Telephone 608-262-5332, FAX 608-262-3453 mtrecord@wisc.edu, rmsaecker@wisc.edu. ¶ These authors contributed equally to this work. 1 Current address: Department of Bacteriology, University of Wisconsin-Madison/Great Lakes Bioenergy Research Center, Madison, WI 53706 Supporting InformationSupporting information reports the irreversible rate constants (k obs ) for formation of open complexes as a function of [RNAP] total and [Na + ] in Cl − buffer (STable 1), and in Glu − and F − buffers (STable 2). STable 3 presents the fits (K heparin , [R] free ) of relaxation to equilibrium data (k r ) at 25 °C to obtain k d in Glu − and F − buffers. This material is available free of charge...

Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free energy to unwind and separate 13 base pairs of λPR promoter DNA to form the unstable open intermediate I2, which rapidly converts to much more stable open complexes (I3, RPo). Conversion of I2 to RPo involves folding/assembly of several mobile RNAP domains on downstream duplex DNA. Here, we investigate effects of a 42-residue deletion in the mobile β’ jaw (ΔJAW) and truncation of promoter DNA beyond +12 (DT+12) on the steps of initiation. We find that in stable ΔJAW open complexes the downstream boundary of hydroxyl radical protection shortens by 5–10 base pairs, as compared to wild-type (WT) complexes. Dissociation kinetics of open complexes formed with ΔJAW RNAP and/or DT+12 DNA resemble those deduced for the structurally-uncharacterized intermediate I3. Overall rate constants (ka) for promoter binding and DNA opening by ΔJAW RNAP are much smaller than for WT RNAP. Values of ka for WT RNAP with DT+12 and full-length λPR are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RPo formation, while downstream DNA functions primarily as the assembly platform after DNA opening.

Bacterial RNA polymerase and a “sigma” transcription factor form an initiation-competent “open” complex at a promoter by disruption of about 14 base pairs. Strand separation is likely initiated at the highly conserved -11 A-T base pair. Amino acids in conserved region 2.3 of the main E. coli sigma factor, σ70, are involved in this process, but their roles are unclear. To monitor the fates of particular bases upon addition of RNA polymerase, promoters bearing single substitutions of the fluorescent A-analog 2-AP at −11 and two other positions in promoter DNA were examined. Evidence was obtained for an open intermediate on the pathway to open complex formation, in which these 2-AP are no longer stacked onto their neighboring bases. The tyrosine at residue 430 in region 2.3 of σ70 was shown to be involved in quenching the fluorescence of a 2-AP substituted at −11, presumably through a stacking interaction. These data refine the structural model for open complex formation and reveal a novel interaction involved in DNA melting by RNA polymerase.

The three-dimensional nature of interactions between enzymes and their substrates often leads to exacting spatial binding orientations and stereoselectivity in chemical catalysis. Dehydrogenases that use NAD + as a redox cofactor tend to show stereospecificity in transferring a hydride to the C4 of the nicotinamide moiety via either the re or the si face. The stereospecificity of this hydride transfer, which results in a prochiral C4 in the reduced NADH, may be determined using deuterated substrates and 1 H NMR spectroscopy. A biochemistry lab activity that combines analysis of the intermolecular interactions and spatial orientation between substrate, cofactor, and enzyme from a recent crystal structure of yeast alcohol dehydrogenase with improved in situ single-tube reaction conditions for elucidating the prochiral specificity of yeast alcohol dehydrogenase through 1 H NMR spectra analysis is presented.