Jayanta Mukhopadhyay, Elena Sineva, Jennifer Knight, Ronald M. Levy and Richard H. Ebright
The antibacterial peptide microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). Biochemical results indicate that inhibition of transcription occurs at the level of NTP uptake or NTP binding by RNAP. Genetic results indicate that inhibition of transcription requires an extensive determinant, comprising more than 50 amino acid residues, within the RNAP secondary channel (also known as the “NTP-uptake channel” or “pore”). Biophysical results indicate that inhibition of transcription involves binding of MccJ25 within the RNAP secondary channel. Molecular modeling indicates that binding of MccJ25 within the RNAP secondary channel obstructs the RNAP secondary channel. We conclude that MccJ25 inhibits transcription by binding within and obstructing the RNAP secondary channel—acting essentially as a “cork in a bottle.” Obstruction of the RNAP secondary channel represents an attractive target for drug discovery.
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Molecular Cell, Volume 33, Issue 1, 16 January 2009, Pages 97-108
Rachel A. Mooney, Sarah E. Davis, Jason M. Peters, Jennifer L. Rowland, Aseem Z. Ansari and Robert Landick
The trafficking patterns of the bacterial regulators of transcript elongation σ70, ρ, NusA, and NusG on genes in vivo and the explanation for promoter-proximal peaks of RNA polymerase (RNAP) are unknown. Genome-wide, E. coli ChIP-chip revealed distinct association patterns of regulators as RNAP transcribes away from promoters (ρ first, then NusA, then NusG). However, the interactions of elongating complexes with these regulators did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from σ70 peaks in the direction of transcription and co-occurred with NusA and ρ peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of ρ did not increase RNAP levels within genes downstream from the RNAP peaks, suggesting the peaks are caused by a mechanism other than ρ-dependent attenuation.
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Molecular Cell, Volume 24, Issue 5, 8 December 2006, Pages 747-757
Nikos B. Reppas, Joseph T. Wade, George M. Church and Kevin Struhl
We perform a genome-wide analysis of the transition between transcriptional initiation and elongation in Escherichia coli by determining the association of core RNA polymerase (RNAP) and the promoter-recognition factor σ70 with respect to RNA transcripts. We identify 1286 σ70-associated promoters, including many internal to known operons, and demonstrate that σ70 is usually released very rapidly from elongating RNAP complexes. On average, RNAP density is higher at the promoter than in the coding sequence, although the ratio is highly variable among different transcribed regions. Strikingly, a significant fraction of RNAP-bound promoters is not associated with transcriptional activity, perhaps due to an intrinsic energetic barrier to promoter escape. Thus, the transition from transcriptional initiation to elongation is highly variable, often rate limiting, and in some cases is essentially blocked such that RNAP is effectively “poised” to transcribe only under the appropriate environmental conditions. The genomic pattern of RNAP density in E. coli differs from that in yeast and mammalian cells.
Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We observe small, but reproducible and abortive-product-length-dependent, decreases in distance between the RNAP leading edge and DNA downstream of RNAP upon abortive initiation, and we observe large decreases in distance upon promoter escape. Inspection of population distributions and single-molecule time traces for abortive initiation indicates that, at a consensus promoter, at saturating ribonucleoside triphosphate concentrations, abortive-product release is rate-limiting (i.e., abortive-product synthesis and RNAP-active-center forward translocation are fast, whereas abortive-product dissociation and RNAP-active-center reverse translocation are slow). The results obtained using this new methodology confirm and extend those obtained from diffusing single molecules, and pave the way for real-time, single-molecule observations of the transitions between various states of the transcription complex throughout transcription.