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 uni®ed 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 b,g-phosphates of the incoming substrate; and during hydrolysis by the phosphates of a non-base-paired nucleoside triphosphate. The latter mode de®nes a transient, non-speci®c nucleoside triphosphate-binding site adjacent to the active center, which may serve as a gateway for polymerization of substrates.

During transcription elongation, RNA polymerase (RNAP) occasionally loses its grip on the growing RNA end and backtracks on the DNA template. Prokaryotic Gre factors rescue the backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes the disengaged 3 RNA segment. By using RNA-protein crosslinking in defined ternary elongating complexes, site-directed mutagenesis, discriminative biochemical assays, and docking of the two protein structures, we show that Gre acts by providing two carboxylate residues for coordination of catalytic Mg 2؉ ion in the RNAP active center. A similar mechanism is suggested for the functionally analogous eukaryotic SII factor. The results expand the general two-metal model of RNAP catalytic mechanism whereby one of the Mg 2؉ ions is permanently retained, whereas the other is recruited ad hoc by an auxiliary factor.catalytic magnesium ͉ Gre proteins P rokaryotic transcription factors GreA and GreB have attracted much attention in the context of their role in the basic mechanism of elongation (1-7). The biochemical activity of Gre proteins is to effect internal cleavage of RNA during transcription elongation. In backtracked ternary elongating complex (TEC), RNA polymerase (RNAP) slides in the reverse direction (8-10), unwinding the DNA͞RNA hybrid and the upstream DNA duplex and extruding a single-stranded 3Ј RNA segment (Fig. 1A). The latter blocks the substrate binding site of the active center (11) so that TEC stalls, but it can be reactivated through the cutting of the disengaged 3Ј RNA segment by nuclease activity intrinsic to the enzyme (12). Gre factors strongly stimulate the cleavage (1); however, their mechanism of action remains unknown.Previously, we suggested a unified catalytic mechanism based on nucleotidyl transfer reaction and involving the same active center for the reactions of phosphodiester bond formation and RNA degradation (13). In our model, the triad of conserved aspartate residues (D460, D462, and D464 in ␤Ј-subunit of RNAP Escherichia coli) coordinates two Mg 2ϩ ions (Fig. 1B), one of which (Mg-I) is permanently retained in the enzyme, whereas the other (Mg-II), is bound transiently and has to be recruited ad hoc for each act of catalysis. In the polymerization reaction, Mg-II is recruited by the incoming substrate; in the exonuclease reaction it is recruited by a noncomplementary NTP; and in pyrophosphorolysis, by pyrophosphate. According to the model, Gre factors could stimulate RNA cleavage by affecting, directly or indirectly, recruitment of Mg-II in the backtracked TEC. Recently Opalka et al. (14) showed that GreB protein binds to RNAP in such a way that its protruding domain could reach the vicinity of the active center via the ''secondary channel'' in the RNAP structure, which normally serves as entry route for NTP substrates. However, the resolution of that analysis was insufficient to decipher precise contacts in TEC so as to distinguish between direct or allosteric effect of Gre. In this article we pre...

Background: Factor-assisted co-transcriptional proofreading and precise selection of NTP substrates provide high transcription fidelity. Results: Both processes can be achieved through active center tuning (ACT) from the inactive to catalytic state in response to establishing recognition contacts of the reaction substrates. Conclusion: High transcription fidelity can be explained by ACT. Significance: Suggested ACT mechanism represents an exceptional example of substrate recognition coupling to catalysis.

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.