initiation ͉ transcription ͉ wrapping ͉ kinetics S pecific transcription initiation by Escherichia coli RNA polymerase (RNAP: core subunit composition ␣ 2 ␤␤Ј ϩ 70 ϭ holoenzyme) at promoter sequences is determined by recognition of DNA (Ϫ10 and Ϫ35 hexamers) upstream of the start site (ϩ1) by the specificity subunit 70 . Subsequent to binding, a series of large-scale conformational changes in both RNAP and promoter DNA create the initiation-competent open complex (RP o ) (1). During these steps, the multisubunit bacterial RNAP acts as an intricate molecular machine and opens Ϸ14 bp of the DNA double helix. Defining the cascade of conformational changes that occur during initiation is essential to understand sequence-and factordependent regulation of the rate of transcription initiation and has important applications in chemical biology and in antibiotic design. However, the intermediates on this pathway are relatively unstable and short-lived and hence are difficult to trap unambiguously. To date, all structural information about complexes known to be on-pathway intermediates in RP o formation has come from chemical and enzymatic DNA footprinting methods.Quantitative kinetic-mechanistic studies find that at least two kinetically significant intermediates, generically designated I 1 and I 2 , precede formation of RP o by E. coli RNAP:where the relatively slow interconversions between I 1 and I 2 are rate-limiting in both the forward and back directions (2, 3). In the mechanism shown in Eq. 1, I 2 and RP o are characterized by their resistance to a short challenge with a polyanionic competitor such as heparin, which acts to sequester any free RNAP present during the challenge. (In contrast, after a 10 to 20 sec challenge with heparin, I 1 complexes, which are in rapid equilibrium with free RNAP and promoter sequences, are eliminated from the population.) Given the high degree of conservation of bacterial RNAP and promoter DNA sequences, this mechanism is likely to describe the key steps in initiation in most prokaryotes. Moreover, conservation of many elements of sequence, structure, and/or function between bacterial and eukaryotic polymerase (pol II) subunits and transcription factors supports the inference that the bacterial intermediates may be homologs of initiation intermediates formed by pol II (4, 5).Recently we (6) and Ross and Gourse (7) found that the presence of DNA upstream of the Ϫ35 promoter recognition hexamer greatly accelerates (up to Ϸ60-fold) the rate-determining isomerization step (conversion of I 1 to I 2 ). Strikingly, DNase I footprinting of I 1 at the strong bacteriophage promoter P R reveals that when nonspecific DNA upstream of base pair Ϫ47 is present, downstream DNA is protected to around ϩ20, and thus bound in the active-site channel of RNAP. However, when DNA upstream of Ϫ47 is deleted,

Binding of activators to upstream DNA sequences regulates transcription initiation by affecting the stability of the initial RNA polymerase (RNAP)-promoter complex and͞or the rate of subsequent conformational changes required to form the open complex (RPO). Here we observe that the presence of nonspecific upstream DNA profoundly affects an early step in formation of the transcription bubble. Kinetic studies with the PR promoter and Escherichia coli RNAP reveal that the presence of DNA upstream of base pair ؊47 greatly increases the rate of forming RPO, without significantly affecting its rate of dissociation. We find that this increase is largely due to an acceleration of the rate-limiting step (isomerization) in RPO formation, a step that occurs after polymerase binds. Footprinting experiments reveal striking structural differences downstream of the transcription start site (؉1) in the first kinetically significant intermediate when upstream DNA is present. On the template strand, the DNase I downstream boundary of this early intermediate is ؉20 when upstream DNA is present but is shortened by approximately two helical turns when upstream DNA beyond ؊47 is removed. KMnO4 footprinting reveals an identical initiation bubble (؊11 to ؉2), but unusual reactivity of template strand upstream cytosines (؊12, ؊14, and ؊15) on the truncated promoter. Based on this work, we propose that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase. Early steps in transcription initiation require interactions with DNA sequences located upstream from the start site (ϩ1). In eukaryotes, assembly of the RNA polymerase (RNAP) II preinitiation complex hinges on the recruitment of the TATAbinding protein to sequences typically located Ϸ25 bp upstream from ϩ1 (see, for example, ref. 1). In Escherichia coli, the specificity factor 70 brings the polymerase machinery to a promoter via interactions with the so-called Ϫ35͞Ϫ10 elements (2, 3). The rates and equilibria of steps involved in binding and opening the DNA are further modulated by proteins (activators, enhancers, and repressors) that bind to sites located even further upstream (1, 4). Strikingly, high-resolution structures of bacterial RNAP and yeast RNAP II reveal a conserved architecture that dictates that upstream interactions cannot place downstream DNA directly in the cleft containing the active site (5-7). Instead, significant DNA deformations must occur to access the cleft. Additionally, the width of the cleft fundamentally determines whether single-or double-stranded DNA can enter. How do interactions with upstream DNA sequences influence these critical steps or other conformational changes that occur in forming the open complex?A paradigmatic example of the role of upstream DNA sequences in regulating transcription is found in the lysis͞lysogeny decision of phage after infection of E. coli (8). DNA sequences separating two divergent ph...

Transcription initiation is a multi-step process involving a series of requisite conformational changes in RNA polymerase (R) and promoter DNA (P) that create the open complex (RP o ). Here we use the small solutes urea and glycine betaine (GB) to probe the extent and type of surface area changes in the formation of RP o between Eσ 70 RNA polymerase and λP R promoter DNA. Effects of urea quantitatively reflect changes in amide surface and are particularly well suited to detect coupled protein folding events. GB provides a qualitative probe for the exposure or burial of anionic surface. Kinetics of formation and dissociation of RP o reveal strikingly large effects of the solutes on the final steps of RP o formation: urea dramatically increases the dissociation rate constant k d , whereas GB decreases the rate of dissociation. Formation of the first kinetically significant intermediate I 1 is disfavored in urea, and moderately favored by GB. GB slows the rate-determining step that converts I 1 to the second kinetically significant intermediate I 2 ; urea has no effect on this step. The most direct interpretation of these data is that recognition of promoter DNA in I 1 involves only limited conformational changes. Notably the data support the following hypotheses: 1) the negatively charged N-terminal domain of σ 70 remains bound in the "jaws" of polymerase in I 1 ; 2) the subsequent rate-determining isomerization step involves ejecting this domain from the jaws, thereby unmasking the active site; and 3) final conversion to RP o involves coupled folding of the mobile downstream clamp of polymerase.Kinetic studies of the process of open complex (RP o ) formation by E. coli RNA polymerase holoenzyme (R: catalytically competent core enzyme, α 2 ββ′ω bound to promoter DNArecognizing σ 70 subunit, also abbreviated Eσ 70 ) at the λP R promoter (P) have demonstrated that a minimum of three steps, involving two kinetically-significant intermediates (I 1 and I 2 ), are required to describe the mechanism (1-4): † This research was supported by NIH grant GM23467 to M. T. R. W. S. K. was supported by NIH Biotechnology Training grant 5 T32 GM08349.* To whom correspondence should be addressed. 433 Babcock Drive, Madison, WI 53706. Email: record@biochem.wisc.edu, rmsaecker@wisc.edu. ∥ These authors contributed equally to this work. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 27. Evidence for at least two kinetically significant intermediates has also been found for open complex formation at the lacUV5 (5), T7A1 (6-9) and λP RM up 1 promoters (10). Although the structures of these intermediates (I 1 , I 2 ) may be promoter-dependent (e.g. the extent to which DNA from -5 to +25 (where +1 is the start site) is protected by the "jaws" of RNAP in I 1 , cf. (9,11-13)), all proposed mechanisms for RP o formation at σ 70 promoters invoke a critical slow (rate-determining) isomerization step after formation of a first kinetically significant intermediate, I 1 . For λP R , this...

Fenamates are a class of nonsteroidal anti-inflammatory drugs (NSAIDs) that are not fully removed during wastewater treatment and can be released to surface waters. Here, near-surface photochemical half-lives were evaluated to range from minutes to hours of four fenamates and the closely related diclofenac. While quantum yields for direct photochemical reactions at the water surface vary widely from 0.071 for diclofenac to <0.001 for mefenamic acid, all fenamates showed significant reactivity towards singlet oxygen and hydroxyl radical with bimolecular reaction rate constants of 1.3-2.8 × 10 M s and 1.1-2.7 × 10 M s, respectively. Photodecay rates increased in the presence of dissolved organic matter (DOM) for diclofenac (+19%), tolfenamic acid (+9%), and mefenamic acid (+95%), but decreased for flufenamic acid (-2%) and meclofenamic acid (-14%) after accounting for light screening effects. Fast reaction rate constants of all NSAIDs with model triplet sensitizers were quantified by laser flash photolysis. Here, the direct observation of diphenylamine radical intermediates by transient absorption spectroscopy demonstrates one-electron oxidation of all fenamates. Quenching rate constants of these radical intermediates by ascorbic acid, a model antioxidant, were also quantified. These observations suggest that the balance of oxidation by photoexcited triplet DOM and quenching of the formed radical intermediates by antioxidant moieties determines whether net sensitization or net quenching by DOM occurs in the photochemical degradation of fenamates.