The σ24 Subunit of Escherichia coli RNA Polymerase Can Induce Transcriptional Pausing in vitro.

The bacterium Escherichia coli has seven σ subunits that bind core RNA polymerase and are necessary for promoter recognition. It was previously shown that the σ70 and σ38 subunits can also interact with the transcription elongation complex (TEC) and stimulate pausing by recognizing DNA sequences similar to the -10 element of promoters. In this study, we analyzed the ability of the σ32, σ28, and σ24 subunits to induce pauses in reconstituted TECs containing corresponding -10 consensus elements. It was found that the σ24 subunit can induce a transcriptional pause depending on the presence of the -10 element. Pause formation is suppressed by the Gre factors, suggesting that the paused complex adopts a backtracked conformation. Some natural promoters contain potential signals of σ24-dependent pauses in the initially transcribed regions, suggesting that such pauses may have regulatory functions in transcription.

[Structural dynamics of the active center of multisubunit RNA polymerases during RNA synthesis and proofreading].

Multisubunit RNA polymerases are complex molecular machines that are responsible for transcription of genes in all cellular organisms and possess several catalytic activities, the most important of which are nucleotide addition to the growing RNA chain and RNA cleavage. Efficient and accurate RNA synthesis requires switching between different RNA polymerase activities that depends on the structural state of the elongation complex and conformational dynamics of the enzyme active center. The RNA polymerase active center contains two magnesium ions, which coordinate reactive groups of substrates, an d structural elements that are involved in the binding a nd correct orientation of substrates and also participate in R NApolymerase translocation. The most important of these elements are G-loop and F-helix and also regions that affect their conformational mobility. In this review, we discussed mechanisms of structural rearrangments taking place in the activecenter of multisubuniit RNA polymerases during transcription and provided several examples of RNA polymerase regulation by factors that affect the binding of catalytic magnesium ions and conformational mobility of G-loop and F-helix.

[Isolation and properties of cysteine protease from Serratia proteamaculans 94].

A new cysteine protease (SpCP) with a molecular mass of about 50 kDa and optimal functioning at pH 8.0 was isolated from the culture medium of a Serratia proteamaculans 94 psychrotolerant strain using affinity and gel permeation chromatography. The enzyme N terminal amino acid sequence (SPVEEAEGDGIVLDV-) exhibits a reliable similarity to N terminal sequences of gingipains R, cysteine proteases from Polphyromonas gingivalis. Unlike gingipains R, SpCP displays a double substrate specificity and cleaves bonds formed by carboxylic groups of Arg, hydrophobic amino acid residues (Val, Leu, Ala, Tyr, and Phe), Pro, and Gly. SpCP can also hydrolyze native collagen. The enzyme catalysis is effective in a wide range of temperatures. Kinetic studies of Z-Ala-Phe-Arg-pNA hydrolysis catalyzed by the protease at 4 and 37 degrees C showed that a decrease in temperature by more than 30 degrees C causes a 1.3-fold increase in the kcat/Km ratio. Thus, SpCP is an enzyme adapted to low positive temperatures. A protease displaying such properties was found in microorganisms of the Serratia genus for the first time and may serve as a virulent factor for these bacteria.

Analysis of RNA cleavage by RNA polymerases from Escherichia coli and Deinococcus radiodurans.

RNA polymerase can both synthesize and cleave RNA. Both reactions occur at the same catalytic center containing two magnesium ions bound to three aspartic acid residues of the absolutely conserved NADFDGD motif of the RNA polymerase beta subunit. We have demonstrated that RNA polymerase from Deinococcus radiodurans possesses much higher rate of intrinsic RNA cleavage than RNA polymerase from Escherichia coli (the difference in the rates is about 15-fold at 20 degrees C). However, these RNA polymerases do not differ in the rates of RNA synthesis. Comparison of the RNA polymerase sequences adjacent to the NADFDGD motif reveals the only amino acid substitution in this region (Glu751 in D. radiodurans vs. Ala455 in E. coli), which is localized in the secondary enzyme channel and can potentially affect the rate of RNA cleavage. Introduction of the corresponding substitution in the E. coli RNA polymerase leads to a slight (about 2-3-fold) increase in the cleavage rate, but does not affect RNA synthesis. Thus, the difference in the RNA cleavage rates between E. coli and D. radiodurans RNA polymerases is likely determined by multiple amino acid substitutions, which do not affect the rate of RNA synthesis and are localized in several regions of the active center.

Cysteine proteinases of microorganisms and viruses.

This review considers properties of secreted cysteine proteinases of protozoa, bacteria, and viruses and presents information on the contemporary taxonomy of cysteine proteinases. Literature data on the structure and physicochemical and enzymatic properties of these enzymes are reviewed. High interest in cysteine proteinases is explained by the discovery of these enzymes mostly in pathogenic organisms. The role of the proteinases in pathogenesis of several severe diseases of human and animals is discussed.