Daria Esyunina

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.

Interplay between the trigger loop and the F loop during RNA polymerase catalysis

The trigger loop (TL) in the RNA polymerase (RNAP) active center plays key roles in the reactions of nucleotide addition and RNA cleavage catalyzed by RNAP. The adjacent F loop (FL) was proposed to contribute to RNAP catalysis by modulating structural changes in the TL. Here, we investigate the interplay between these two elements during transcription by bacterial RNAP. Thermodynamic analysis of catalysis by RNAP variants with mutations in the TL and FL suggests that the TL is the key element required for temperature activation in RNAP catalysis, and that the FL promotes TL transitions during nucleotide addition. We reveal characteristic differences in the catalytic parameters between thermophilic Thermus aquaticus and mesophilic Deinococcus radiodurans RNAPs and identify the FL as an adaptable element responsible for the observed differеnces. Mutations in the FL also significantly affect the rate of intrinsic RNA cleavage in a TL-dependent manner. In contrast, much weaker effects of the FL and TL mutations on GreA-assisted RNA cleavage suggest that the FL-dependent TL transitions are not required for this reaction. Thus, functional interplay between the FL and TL is essential for various catalytic activities of RNAP and plays an adaptive role in catalysis by thermophilic and mesophilic enzymes.

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.

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.

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Significance Transcription termination is an essential regulatory step in gene expression, but its molecular mechanisms remain less understood in comparison with other steps of transcription, owing to the highly dynamic nature of the termination process. Antitermination factors play important regulatory roles in bacteria, viruses, and eukaryotes, but only a few such factors have been studied in molecular detail. We describe a pathway of transcription antitermination by a viral factor that involves suppression of RNA polymerase pausing and stabilization of the transcription complex through interactions with an evolutionary conserved ω subunit of RNA polymerase. The results of the study extend the list of universal RNA polymerase sites able to accept regulatory inputs and provide new insights into the mechanisms of transcription termination. Transcription antitermination is a common strategy of gene expression regulation, but only a few transcription antitermination factors have been studied in detail. Here, we dissect the transcription antitermination mechanism of Xanthomonas oryzae virus Xp10 protein p7, which binds host RNA polymerase (RNAP) and regulates both transcription initiation and termination. We show that p7 suppresses intrinsic termination by decreasing RNAP pausing and increasing the transcription complex stability, in cooperation with host-encoded factor NusA. Uniquely, the antitermination activity of p7 depends on the ω subunit of the RNAP core and is modulated by ppGpp. In contrast, the inhibition of transcription initiation by p7 does not require ω but depends on other RNAP sites. Our results suggest that p7, a bifunctional transcription factor, uses distinct mechanisms to control different steps of transcription. We propose that regulatory functions of the ω subunit revealed by our analysis may extend to its homologs in eukaryotic RNAPs.

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Significance Tight and highly controlled transcriptional regulation is pivotal for cell survival under stress conditions. We demonstrate that Gre factor homolog (Gfh) proteins found in the Deinococcus/Thermus lineage of extremophilic bacteria affect multiple steps of transcription and may serve to halt transcription at specific genomic sites. These proteins bind within the secondary channel of RNA polymerase (RNAP) and dramatically increase site-specific transcriptional pausing in the presence of manganese ions, likely by stabilizing inactive enzyme conformation. Both Gfh expression and cellular manganese concentration are increased during stress response, thus providing an efficient way for transcription regulation. Similar mechanisms may be operating with other prokaryotic and eukaryotic factors acting through the secondary channel, which emerges as the central regulatory hub in the control of RNAP activities. Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stress-resistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg2+ or Mn2+) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.

Possible roles of σ-dependent RNA polymerase pausing in transcription regulation

ABSTRACT The σ subunit of bacterial RNA polymerase is required for promoter recognition during transcription initiation but may also regulate transcription elongation. The principal σ70 subunit of Escherichia coli was shown to travel with RNA polymerase and induce transcriptional pausing at promoter-like motifs, with potential regulatory output. We recently demonstrated that an alternative σ38 subunit can also induce RNA polymerase pausing. Here, we outline proposed regulatory roles of σ-dependent pausing in bacteria and discuss possible interplay between alternative σ variants and regulatory factors during transcription elongation.

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

Lineage‐specific Gfh factors from the radioresistant bacterium Deinococcus radiodurans, which bind within the secondary channel of RNA polymerase, stimulate transcriptional pausing at a wide range of pause signals (elemental, hairpin‐dependent, post‐translocated, backtracking‐dependent, and consensus pauses) and increase intrinsic termination. Universal bacterial factor NusA, which binds near the RNA exit channel, enhances the effects of Gfh factors on termination and hairpin‐dependent pausing but do not act on other pause sites. It is proposed that NusA and Gfh target different steps in the pausing pathway and may act together to regulate transcription under stress conditions. Thus, transcription factors that interact with nascent RNA in the RNA exit channel can communicate with secondary channel regulators to modulate RNA polymerase activities.

σ38-dependent promoter-proximal pausing by bacterial RNA polymerase

Abstract Transcription initiation by bacterial RNA polymerase (RNAP) requires a variable σ subunit that directs it to promoters for site-specific priming of RNA synthesis. The principal σ subunit responsible for expression of house-keeping genes can bind the transcription elongation complex after initiation and induce RNAP pausing through specific interactions with promoter-like motifs in transcribed DNA. We show that the stationary phase and stress response σ38 subunit can also induce pausing by Escherichia coli RNAP on DNA templates containing promoter-like motifs in the transcribed regions. The pausing depends on σ38 contacts with the DNA template and RNAP core enzyme and results in formation of backtracked transcription elongation complexes, which can be reactivated by Gre factors that induce RNA cleavage by RNAP. Our data suggest that σ38 can bind the transcription elongation complex in trans but likely acts in cis during transcription initiation, by staying bound to RNAP and recognizing promoter-proximal pause signals. Analysis of σ38-dependent promoters reveals that a substantial fraction of them contain potential pause-inducing motifs, suggesting that σ38-depended pausing may be a common phenomenon in bacterial transcription.

Single-strand promoter traps for bacterial RNA polymerase.

Besides canonical double-strand DNA promoters, multisubunit RNAPs (RNA polymerases) recognize a number of specific single-strand DNA and RNA templates, resulting in synthesis of various types of RNA transcripts. The general recognition principles and the mechanisms of transcription initiation on these templates are not fully understood. To investigate further the molecular mechanisms underlying the transcription of single-strand templates by bacterial RNAP, we selected high-affinity single-strand DNA aptamers that are specifically bound by RNAP holoenzyme, and characterized a novel class of aptamer-based transcription templates. The aptamer templates have a hairpin structure that mimics the upstream part of the open promoter bubble with accordingly placed specific promoter elements. The affinity of the RNAP holoenzyme to such DNA structures probably underlies its promoter-melting activity. Depending on the template structure, the aptamer templates can direct synthesis of productive RNA transcripts or effectively trap RNAP in the process of abortive synthesis, involving DNA scrunching, and competitively inhibit promoter recognition. The aptamer templates provide a novel tool for structure-function studies of transcription initiation by bacterial RNAP and its inhibition.