Danil Pupov

Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme.

Background: Cellular RNA polymerases start transcription by de novo RNA priming. Results: Structures and biochemical studies of initially transcribing complexes elucidate the de novo transcription initiation and early stage of RNA transcription. Conclusion: 5′-end of RNA in the transcribing complex starts σ ejection from core enzyme. Significance: Insights from this study can be applicable to all cellular RNA polymerases. The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5′-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.

Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape.

The σ subunit of bacterial RNA polymerase (RNAP) has been implicated in all steps of transcription initiation, including promoter recognition and opening, priming of RNA synthesis, abortive initiation and promoter escape. The post-promoter-recognition σ functions were proposed to depend on its conserved region σ3.2 that directly contacts promoter DNA immediately upstream of the RNAP active centre and occupies the RNA exit path. Analysis of the transcription effects of substitutions and deletions in this region in Escherichia coli σ70 subunit, performed in this work, suggests that (i) individual residues in the σ3.2 finger collectively contribute to RNA priming by RNAP, likely by the positioning of the template DNA strand in the active centre, but are not critical to promoter escape; (ii) the physical presence of σ3.2 in the RNA exit channel is important for promoter escape; (iii) σ3.2 promotes σ dissociation during initiation and suppresses σ-dependent promoter-proximal pausing; (iv) σ3.2 contributes to allosteric inhibition of the initiating NTP binding by rifamycins. Thus, region σ3.2 performs distinct functions in transcription initiation and its inhibition by antibiotics. The B-reader element of eukaryotic factor TFIIB likely plays similar roles in RNAPII transcription, revealing common principles in transcription initiation in various domains of life.

Multiple roles of the RNA polymerase β′ SW2 region in transcription initiation, promoter escape, and RNA elongation

Interactions of RNA polymerase (RNAP) with nucleic acids must be tightly controlled to ensure precise and processive RNA synthesis. The RNAP β′-subunit Switch-2 (SW2) region is part of a protein network that connects the clamp domain with the RNAP body and mediates opening and closing of the active center cleft. SW2 interacts with the template DNA near the RNAP active center and is a target for antibiotics that block DNA melting during initiation. Here, we show that substitutions of a conserved Arg339 residue in the Escherichia coli RNAP SW2 confer diverse effects on transcription that include defects in DNA melting in promoter complexes, decreased stability of RNAP/promoter complexes, increased apparent KM for initiating nucleotide substrates (2- to 13-fold for different substitutions), decreased efficiency of promoter escape, and decreased stability of elongation complexes. We propose that interactions of Arg339 with DNA directly stabilize transcription complexes to promote stable closure of the clamp domain around nucleic acids. During initiation, SW2 may cooperate with the σ3.2 region to stabilize the template DNA strand in the RNAP active site. Together, our data suggest that SW2 may serve as a key regulatory element that affects transcription initiation and RNAP processivity through controlling RNAP/DNA template interactions.

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.

Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases

RNA cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in bacterial stress adaptation.

Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription

During transcription, the catalytic core of RNA polymerase (RNAP) must interact with the DNA template with low-sequence specificity to ensure efficient enzyme translocation and RNA extension. Unexpectedly, recent structural studies of bacterial promoter complexes revealed specific interactions between the nontemplate DNA strand at the downstream edge of the transcription bubble (CRE, core recognition element) and a protein pocket formed by core RNAP (CRE pocket). We investigated the roles of these interactions in transcription by analyzing point amino acid substitutions and deletions in Escherichia coli RNAP. The mutations affected multiple steps of transcription, including promoter recognition, RNA elongation and termination. In particular, we showed that interactions of the CRE pocket with a nontemplate guanine immediately downstream of the active center stimulate RNA-hairpin-dependent transcription pausing but not other types of pausing. Thus, conformational changes of the elongation complex induced by nascent RNA can modulate CRE effects on transcription. The results highlight the roles of specific core RNAP–DNA interactions at different steps of RNA synthesis and suggest their importance for transcription regulation in various organisms.

Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation

Background: RNA polymerases (RNAPs) from Thermus aquaticus and Escherichia coli differ in many aspects of transcription initiation. Results: Regions 1.1 and 1.2 of the σ subunit determine instability and cold sensitivity of promoter complexes of T. aquaticus RNAP. Conclusion: Substitutions in σ regions 1.1 and 1.2 modulate RNAP-promoter interactions. Significance: Evolutionary changes in the σ subunit determine functional differences between bacterial RNAPs during transcription initiation. RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ70 and T. aquaticus σA subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ70 subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the −10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.

Isolation and properties of Serratia proteamaculans 94 cysteine protease

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 Porphyromonas 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°C showed that a decrease in temperature by more than 30°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 β′ 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°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.

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

Multisubunit RNA polymerases (RNAPs) 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. Switching between different RNAP activities is essential for efficient and accurate RNA synthesis and depends on the structural state of the elongation complex and the conformational dynamics of the active center of the enzyme. The active center of RNAP contains two magnesium ions, which coordinate the reactive groups of substrates, and structural elements that are involved in the binding and correct orientation of substrates and in RNAP translocation. The most important of these elements are G-loop, F-helix, and regions that affect their conformational mobility. The review discusses the mechanisms of structural rearrangements that take place in the active center of RNAP during transcription. Several examples are provided for the RNAP regulation by factors that affect the binding of the catalytic magnesium ions and the conformational mobility of the G-loop and F-helix.