Structural organization of the RNA polymerase-promoter open complex.

Naryshkin, N, Revyakin A, Kim Y, Mekler V, Ebright RH. 2000.

Journal:

Cell

Volume Number:

101

Issue Number:

6

Pages:

601-11

Abstract:

We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.

Citation:: Naryshkin, N, Revyakin A, Kim Y, Mekler V, Ebright RH. 2000. Structural organization of the RNA polymerase-promoter open complex.. Cell. 101(6):601-11.

Dr. Richard H. Ebright

Principal Investigator

Recent Publications

Robb, NC, Cordes T, Hwang L C, Gryte K, Duchi D, Craggs TD, Santoso Y, Weiss S, Ebright RH, Kapanidis AN. 2013. The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.. Journal of molecular biology. 425:875-885. Abstract

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 base-pairs around the transcription start site, and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts, and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule Förster Resonance Energy Transfer. We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labelling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

Zhang, Y, Feng Y, Chatterjee S, Tuske S, Ho MX, Arnold E, Ebright RH. 2012. Structural Basis of Transcription Initiation.. Science (New York, N.Y.). 338(6110):1076-1080. AbstractWebsite

During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, σ(A), and a promoter DNA fragment corresponding to the transcription bubble and downstream dsDNA of the RNAP-promoter open complex. The structures show that σ recognizes the -10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases, and that RNAP recognizes the -4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between σ and template-strand ssDNA preorganize template-strand ssDNA to engage the RNAP active center.

Srivastava, A, Degen D, Ebright YW, Ebright RH. 2012. Frequency, Spectrum, and Nonzero Fitness Costs of Resistance to Myxopyronin in Staphylococcus aureus.. Antimicrobial agents and chemotherapy. 56(12):6250-5. Abstract

The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx-the RNAP "switch region SW1/SW2 subregion"-is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here, we report the frequency, spectrum, and fitness costs of Myx resistance in Staphylococcus aureus. The resistance rate for Myx is 4 × 10(-8) to 7 × 10(-8) per generation, which is equal within error to the resistance rate for Rif (3 × 10(-8) to 10 × 10(-8) per generation). Substitutions conferring Myx resistance were obtained in the RNAP β subunit [six substitutions: V1080(1275)I, V1080(1275)L, E1084(1279)K, D1101(1296)E, S1127(1322)L, and S1127(1322)P] and the RNAP β' subunit [five substitutions: K334(345)N, T925(917)K, T925(917)R, G1172(1354)C, and G1172(1354)D] (residues numbered as in Staphylococcus aureus RNAP and, in parentheses, as in Escherichia coli RNAP). Sites of substitutions conferring Myx resistance map to the RNAP switch region SW1/SW2 subregion and do not overlap the binding site on RNAP for Rif, and, correspondingly, Myx-resistant mutants exhibit no cross-resistance to Rif. All substitutions conferring Myx resistance exhibit significant fitness costs (4 to 15% per generation). In contrast, at least three substitutions conferring Rif resistance exhibit no fitness costs (≤0% per generation). The observation that all Myx-resistant mutants have significant fitness costs whereas at least three Rif-resistant mutants have no fitness costs, together with the previously established inverse correlation between fitness cost and clinical prevalence, suggests that Myx resistance is likely to have lower clinical prevalence than Rif resistance.

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