To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes, and not from abortive initiation. Analysis of productiveinitiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than ten-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4-and 5-mer, 7-mer to 9-mer and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously-determined 9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of incleft, −10 and −35 interactions. Parallels to escape by T7 RNAP are discussed.
Advances in cryo-electron microscopy have opened up new avenues to structurally define biomolecular assemblies. To arrive at detailed psedoatomic models, it is necessary to employ integrative computational modeling. Here,
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.
The lipid bilayer in a cell membrane serves as a natural solvent for membrane proteins. The physical principle of how the lipid bilayer mediates the folding, stability, and cooperativity between residues to facilitate the functions of membrane proteins in cells is not well understood. Here, we employed steric trapping as a tool to study membrane protein folding in a native lipid environment. This method couples spontaneous denaturation of a doubly biotinylated protein to the competitive binding of bulky monovalent streptavidin. We determined the thermodynamic stability (DG o N-D) of the intramembrane protease GlpG of Escherichia coli and analyzed the side chain contribution to the stability at 37 residue sites in both lipid bilayers and detergent micelles. We find that the lipid bilayer stabilizes GlpG in several distinct ways in comparison to micelles: (1)The lipid bilayer enhances GlpG stability by 1.6-1.9 kcal/mol; (2) Mutations of buried residues induce larger deleterious effects on the stability in the bilayer by up to 1.5-to 1.9-fold in DDG o N-D, WT-Mut ; (3) Our cooperativity profiling analysis reveals that, while the cooperative side-chain interactions are clustered in a few defined regions of the protein interior in micelles, they become dominant throughout all packed regions in the bilayer. This result demonstrates that the lipid bilayer not only serves as a hydrophobic medium that surrounds membrane proteins but also substantially impacts the strengths of side-chain packing and their interaction network.
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