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Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription., RNA synthesis by bacterial RNA polymerase is interrupted by pauses but their role in RNA synthesis is poorly understood. Here the authors use single-molecule FRET and biochemical analysis to show that pausing regulates branching between the abortive and productive outcomes of initial transcription.

International audience; The s subunit of bacterial RNA polymerase (RNAP) controls recognition of the −10 and −35 promoter elements during transcription initiation. Free s adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of s activation proposes that binding to core RNAP induces formation of an "open," active, s conformation, which is optimal for promoter recognition. Using single-molecule Förster resonance energy transfer, we demonstrate that vegetative-type s subunits exist in open and closed states even after binding to the RNAP core. As an extreme case, RNAP from Mycobacterium tuberculosis preferentially retains s in the closed conformation, which is converted to the open conformation only upon binding by the activator protein RbpA and interaction with promoter DNA. These findings reveal that the conformational dynamics of the s subunit in the RNAP holoenzyme is a target for regulation by transcription factors and plays a critical role in promoter recognition.

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

International audience; Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification of clinically relevant regulators is needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts by regulating actin dynamics. This is of particular importance since actin cytoskeleton is essential to generate the sealing zone, an osteoclast-specific structure ultimately mediating bone resorption. Here, we report that the atypical Rac1 exchange factor Dock5 is necessary for osteoclast function both in vitro and in vivo. We uncovered that establishment of the sealing zone and consequently osteoclast resorbing activity in vitro require Dock5. Mechanistically, our results suggest that osteoclasts lacking Dock5 have impaired adhesion that can be explained by perturbed Rac1 and p130Cas activities. Consistent with these functional assays, we identified a novel small molecule inhibitor of Dock5 capable of hindering osteoclast resorbing activity. To investigate the in vivo relevance of these findings, we studied Dock5-/- mice and uncovered that they have increased trabecular bone mass, with normal osteoclast numbers, confirming that Dock5 is essential for bone resorption but not for osteoclast differentiation. Taken together, our findings characterize Dock5 as a regulator of osteoclast function and as a potential novel target to develop anti-osteoporotic treatments. © 2010 American Society for Bone and Mineral Research.

International audience; Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification of clinically relevant regulators is needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts by regulating actin dynamics. This is of particular importance since actin cytoskeleton is essential to generate the sealing zone, an osteoclast-specific structure ultimately mediating bone resorption. Here, we report that the atypical Rac1 exchange factor Dock5 is necessary for osteoclast function both in vitro and in vivo. We uncovered that establishment of the sealing zone and consequently osteoclast resorbing activity in vitro require Dock5. Mechanistically, our results suggest that osteoclasts lacking Dock5 have impaired adhesion that can be explained by perturbed Rac1 and p130Cas activities. Consistent with these functional assays, we identified a novel small molecule inhibitor of Dock5 capable of hindering osteoclast resorbing activity. To investigate the in vivo relevance of these findings, we studied Dock5-/- mice and uncovered that they have increased trabecular bone mass, with normal osteoclast numbers, confirming that Dock5 is essential for bone resorption but not for osteoclast differentiation. Taken together, our findings characterize Dock5 as a regulator of osteoclast function and as a potential novel target to develop anti-osteoporotic treatments. © 2010 American Society for Bone and Mineral Research.