Initiation of transcription by E. coli RNA polymerase (RNAP) begins with specific binding to promoter DNA and ends with promoter escape. For the four T7A1 and λPR promoter/discriminator combinations, we found that the discriminator determined the lifetime and stability of the open complex and the escape point of RNAP in initiation from productive promoter complexes (1). Quantitative initiation studies with these promoter/discriminator combinations reveal that the RNA‐DNA hybrid length for promoter escape and the length distribution of short (abortive) RNA released before escape both increase with the lifetime or stability of the initiation‐competent open complex (OC). The escape point for productive complexes increases from the 7‐mer to 8‐mer step for T7A1 discriminators to the 10‐mer to 11‐mer step for λPR discriminators. Nonproductive complexes make a short RNA smaller than the escape length and stall, slowly releasing it and reinitiating. These findings led us to predict that escape from a promoter like the ribosomal rrnB P1 promoter, with an unstable OC, should occur at a very short RNA‐DNA hybrid length and without release of short RNAs (1). Currently, to obtain the experimental data to test this prediction, we are performing single round transcription assays for the rrnB P1 promoter and other promoters with the rrnB P1 discriminator. In preliminary studies, we have found NTP concentrations where a significant fraction of promoters initiate rapidly. For these NTP concentrations, we find that only the shortest RNAs (e.g. 3‐mer) are made by nonproductive complexes during the time period required for productive complexes to escape and begin elongation. In addition, initiation of λPR promoter with rrnB P1 discriminator shows similar pattern comparing to initiation of rrnB P1 promoter with its own discriminator. We conclude that the current results are consistent with the proposal of early escape of RNAP from promoters with unstable OC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
complex (PIC). After PIC formation, double-stranded DNA is unwound to form a single-stranded DNA bubble and the template strand is loaded into the polymerase active site. Initial DNA opening is ATP-dependent and is catalyzed by Ssl2/XPB, a dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase where downstream DNA is searched for optimal start-sites. Here, to test different models for initial DNA opening and start-site scanning, we measure the size of the DNA bubble formed by Saccharomyces cerevisiae PICs in real time using a single-molecule magnetic tweezers assay. We show that ATP hydrolysis by Ssl2 leads to the opening of a 6 base-pair (bp) DNA bubble that is expanded to 13 bp in the presence of NTPs. These observations support a two-step DNA opening model wherein ATPdependent Ssl2 translocation leads to a 6 bp open complex which RNA polymerase II expands via NTP-dependent RNA transcription.
The nucleoprotein from the measles virus (MeV) possesses a C-terminal intrinsically disordered domain, referred to as N TAIL , which is essential for virus replication. N TAIL binds to the folded X domain of phosphoprotein P (P XD ), enabling recruitment of the polymerase complex and fine regulation of viral messenger synthesis. N TAIL is highly disordered in the unbound state, and partially folds upon binding to P XD . However, the majority of N TAIL remains disordered in the bound state. The folding upon binding of N TAIL has been characterized using a large number of experimental techniques, revealing many important aspects of binding. However, the role of dynamical reconfiguration of N TAIL disordered regions, and the mechanism by which these contribute to binding remains elusive. To characterize the large-scale dynamical reconfigurations of N TAIL in solution, in the bound and unbound states, we use photo-induced electron transfer (PET) between tryptophan (W) and cysteine (C). This technique allows probing both the W-C distance and the rate of W-C contact formation in IDPs, without using prosthetic dyes. Measured PET rates can be directly compared to results from molecular simulations, providing a more detailed description of IDP conformational ensembles and dynamics. By generating a series of N TAIL W-C mutants, we use PET to probe different regions of the protein, and their modifications upon binding. We compare our experimental results to molecular simulations of N TAIL in the free and bound states. The combined results provide insights into the role of disordered regions, and their conformational dynamics in IDP binding.
Translation requires efficient peptidyl transfer to support the cellular needs. Of the factors that facilitate the peptide elongation, elongation factor Tu (EF-Tu) is crucial in helping the correct aminoacyl-tRNA to be incorporated into the protein. It also acts as an energy carrier to move aminoacyl-tRNA near to P site peptides, forming a new peptide bond. Here, we investigated diffusive motions of translationally fused EF-Tu (TufA and TufB) with mEos2 via single particle tracking photoactivation localization microscopy (spt-PALM) on live E. coli cells. The mean diffusion coefficient of EF-Tu tracked at 100 Hz was 0.15 um^2/s. The spatial distribution of EF-Tu is reminiscent of that of translating ribosomes and its diffusion coefficient is five times faster than 70S diffusion (0.03 um^2/s). However, this is still much slower compared to the expected diffusion of free EF-Tu-size proteins such as GFP (5 um^2/s). This suggests that EF-Tu spends most of the time on ribosome or/and on a large complex. We will further investigate the cause of slow diffusive motion via antibiotic treatments and mutations on translation machinery.
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