The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase

Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg(2+) ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg(2+) ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the β′ subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg(2+) binding to free RNAP below detection limits, the apparent affinity to Mg(2+) in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg(2+)

Structural transitions in the transcription elongation complexes of bacterial RNA polymerase during σ-dependent pausing

A transcription initiation factor, the σ70 subunit of Escherichia coli RNA polymerase (RNAP) induces transcription pausing through the binding to a promoter-like pause-inducing sequence in the DNA template during transcription elongation. Here, we investigated the mechanism of σ-dependent pausing using reconstituted transcription elongation complexes which allowed highly efficient and precisely controlled pause formation. We demonstrated that, following engagement of the σ subunit to the pause site, RNAP continues RNA synthesis leading to formation of stressed elongation complexes, in which the nascent RNA remains resistant to Gre-induced cleavage while the transcription bubble and RNAP footprint on the DNA template extend in downstream direction, likely accompanied by DNA scrunching. The stressed complexes can then either break σ-mediated contacts and continue elongation or isomerize to a backtracked conformation. Suppressing of the RNAP backtracking decreases pausing and increases productive elongation. On the contrary, core RNAP mutations that impair RNAP interactions with the downstream part of the DNA template stimulate pausing, presumably by destabilizing the stressed complexes. We propose that interplay between DNA scrunching and RNAP backtracking may have an essential role in transcription pausing and its regulation in various systems

Mesenchymal Stem Cells and Myeloid Derived Suppressor Cells: Common Traits in Immune Regulation

To protect host against immune-mediated damage, immune responses are tightly regulated. The regulation of immune responses is mediated by various populations of mature immune cells, such as T regulatory cells and B regulatory cells, but also by immature cells of different origins. In this review, we discuss regulatory properties and mechanisms whereby two distinct populations of immature cells, mesenchymal stem cells, and myeloid derived suppressor cells mediate immune regulation, focusing on their similarities, discrepancies, and potential clinical applications

Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase

In DNA-dependent RNA polymerases, reactions of RNA synthesis and degradation are performed by the same active center (in contrast to DNA polymerases in which they are separate). We propose a unified catalytic mechanism for multisubunit RNA polymerases based on the analysis of its 3′–5′ exonuclease reaction in the context of crystal structure. The active center involves a symmetrical pair of Mg(2+) ions that switch roles in synthesis and degradation. One ion is retained permanently and the other is recruited ad hoc for each act of catalysis. The weakly bound Mg(2+) is stabilized in the active center in different modes depending on the type of reaction: during synthesis by the β,γ-phosphates of the incoming substrate; and during hydrolysis by the phosphates of a non-base-paired nucleoside triphosphate. The latter mode defines a transient, non-specific nucleoside triphosphate-binding site adjacent to the active center, which may serve as a gateway for polymerization of substrates

Antigen-Specific IFN-γ Responses Correlate with the Activity of M. tuberculosis Infection but Are Not Associated with the Severity of Tuberculosis Disease

IFN-γ is a key cytokine in antituberculosis (TB) defense. However, how the levels of its secretion affect M. tuberculosis (Mtb) infection is not clear. We have analyzed associations between IFN-γ responses measured in QuantiFERON®-TB Gold In-tube (QFT) assay, TB disease severity, and Mtb infection activity. TB severity was evaluated based on the results of radiological, microbiological, and clinical examinations. Antigen-driven IFN-γ secretion did not correlate with TB severity. Mitogen-induced IFN-γ secretion correlated inversely with the form of pulmonary pathology and the area of affected pulmonary tissue; the levels of spontaneous IFN-γ secretion correlated with patients’ age (r = 0.395, p = 0.001). Mtb infection activity was evaluated based on radiological data of lung tissue infiltration, destruction, dissemination or calcification, and condensation. The rate of positive QFT results and the levels of antigen-driven IFN-γ secretion increased in a row: patients with residual TB lesions < patients with low TB activity < patients with high TB activity. Thus, antigen-driven IFN-γ secretion and QFT results did not associate with TB severity but associated with the infection activity. The results suggest that quantitative parameters of IFN-γ secretion play a minor role in determining the course of TB disease but mirror the activity of the infectious process