Abstract
Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ(70) from Escherichia coli, the β'-σ complex, and the σ(70) RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ(70), free or in the context of the holoenzyme, recognizes the -10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the -10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the -10 element. We further show that region 1 of σ(70) is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β' RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended -10 promoter element by σ(70). The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.
Highlights
Original recognition must proceed in the form of double-stranded DNA, in transcription-competent open complex, the Ϫ10 element is present in single-stranded form as part of the transcription bubble, which on most promoters extends from Ϫ12 to approximately ϩ3
We reasoned that the interaction of the E. coli RNA polymerase (RNAP) 70 subunit region 2 with the Ϫ10 promoter element might lend itself to such an assay because it is known that this interaction involves multiple aromatic amino acids of 70 region 2.3 that change their environment upon interaction with DNA [27,28,29]
The assay reports only on protein-DNA interactions confined to small parts of RNAP and DNA adjacent to 70 region 2.3 aromatic residues, i.e. on specific interactions important for promoter complex formation
Summary
Materials—Oligodeoxynucleotides were synthesized by Integrated DNA Technologies. Tetramethylrhodamine-5maleimide (TMR), BODIPY FL N-(2-aminoethyl)maleimide were purchased from Invitrogen, and ATTO 520-maleimide was purchased from ATTO-TEC. Final assay mixtures (800 l) contained 1 nM labeled 70 or RNAP holoenzyme and DNA probes at various concentrations. Data Analysis—To obtain equilibrium dissociation constants (Kd), the experimental dependence of the fluorescent signal amplitude (F) on DNA probe concentration (C) was fit to Equation 1,. Oligo probes bearing mutations at Ϫ7 and Ϫ11 positions produced very low fluorescent signal Dissociation constants of these probes were obtained from competition binding experiments using the consensus single-stranded probe as a ref-. Equation 2 is valid at C0 ϾϾ [RNAP], a condition that was fulfilled in our experiments Another equilibrium competition binding assay was used to measure affinity of tight RNAP-fork junction complexes (Kd value lower than 0.1 nM). A double-stranded (Ϫ58/Ϫ14) probe (shown in Fig. 4) producing negligible signal upon binding to (211Cys-TMR) 70 holo-RNAP was used as a competitor. Typical kinetics of reaching equilibrium degree of saturation of RNAP with the (Ϫ38/Ϫ3)(Ϫ38/Ϫ12)Ϫ35mut and (Ϫ58/Ϫ14) probes is shown in supplemental Fig. S3
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