Sergei Borukhov

Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase

Prokaryotic transcription elongation factors GreA and GreB stimulate intrinsic nucleolytic activity of RNA polymerase (RNAP). The proposed biological role of Gre‐induced RNA hydrolysis includes transcription proofreading, suppression of transcriptional pausing and arrest, and facilitation of RNAP transition from transcription initiation to transcription elongation. Using an array of biochemical and molecular genetic methods, we mapped the interaction interface between Gre and RNAP and identified the key residues in Gre responsible for induction of nucleolytic activity in RNAP. We propose a structural model in which the C‐terminal globular domain of Gre binds near the opening of the RNAP secondary channel, the N‐terminal coiled‐coil domain (NTD) protrudes inside the RNAP channel, and the tip of the NTD is brought to the immediate vicinity of RNAP catalytic center. Two conserved acidic residues D41 and E44 located at the tip of the NTD assist RNAP by coordinating the Mg2+ ion and water molecule required for catalysis of RNA hydrolysis. If so, Gre would be the first transcription factor known to directly participate in the catalytic act of RNAP.

Transcription through the roadblocks: the role of RNA polymerase cooperation

During transcription, cellular RNA polymerases (RNAP) have to deal with numerous potential roadblocks imposed by various DNA binding proteins. Many such proteins partially or completely interrupt a single round of RNA chain elongation in vitro. Here we demonstrate that Escherichia coli RNAP can effectively read through the site‐specific DNA‐binding proteins in vitro and in vivo if more than one RNAP molecule is allowed to initiate from the same promoter. The anti‐roadblock activity of the trailing RNAP does not require transcript cleavage activity but relies on forward translocation of roadblocked complexes. These results support a cooperation model of transcription whereby RNAP molecules behave as ‘partners’ helping one another to traverse intrinsic and extrinsic obstacles.

Bacterial transcription elongation factors: new insights into molecular mechanism of action

Like transcription initiation, the elongation and termination stages of transcription cycle serve as important targets for regulatory factors in prokaryotic cells. In this review, we discuss the recent progress in structural and biochemical studies of three evolutionarily conserved elongation factors, GreA, NusA and Mfd. These factors affect RNA polymerase (RNAP) processivity by modulating transcription pausing, arrest, termination or anti‐termination. With structural information now available for RNAP and models of ternary elongation complexes, the interaction between these factors and RNAP can be modelled, and possible molecular mechanisms of their action can be inferred. The models suggest that these factors interact with RNAP at or near its three major, nucleic acid‐binding channels: Mfd near the upstream opening of the primary (DNA‐binding) channel, NusA in the vicinity of both the primary channel and the RNA exit channel, and GreA within the secondary (backtracked RNA‐binding) channel, and support the view that these channels are involved in the maintenance of RNAP processivity.

Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase.

Publisher Summary This chapter discusses the use of histidine (His) tags for obtaining transcription intermediates. The mechanism and regulation of transcription depends largely on the development of experimental techniques permitting dissection of the multistep transcriptional cycle. The His tag technology has been applied for the study of mechanisms of elongation, pausing, factor-independent termination, and interaction of RNA polymerase (RNAP) with transcriptional factors. In addition, histidine tags have been used for the rapid purification of RNAP from cells, in vitro reconstitution of RNAP from individually expressed subunits, screening of genetically engineered RNAP mutations, identification of specific fragments among the products of partial proteolysis, and probing of the surface of a RNAP molecule. The chapter discusses the preparation of His-tagged RNAP from cells. Solid-phase transcription with His-tagged RNAP is also given.

Role of the RNA polymerase sigma subunit in transcription initiation

In bacteria, sigma subunits direct the catalytically competent RNA polymerase core enzyme to promoters. Recent advances in our understanding of bacterial RNA polymerase reveal that sigma subunits are intimately involved in all aspects of transcription initiation including promoter location, promoter melting, initiation of RNA synthesis, abortive initiation and promoter escape.

Analysis of Promoter Targets for Escherichia coli Transcription Elongation Factor GreA In Vivo and In Vitro

Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of Escherichia coli strains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.

Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE

The strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc‐NADH dehydrogenase and the Nar‐nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen‐sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase σA holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.

Biochemical Assays of Gre Factors of Thermus Thermophilus

Publisher Summary This chapter discusses the biochemical assaying of Gre factors of Thermus Thermophilus. The biochemical studies of Escherichia coli Gre factors indicate that they are not nucleases but RNAP co-factors, which activate the same catalytic center involved in both RNA synthesis and RNA hydrolysis reactions. The biological role of factor-induced endonucleolytic reaction includes: the enhancement of transcription fidelity, by helping RNAP excise misincorporated nucleotides; suppression of transcriptional pausing and arrest by reactivation of RNAP during reversible and irreversible backtracking; and stimulation of RNAP promoter escape and transition from initiation to elongation stage of transcription by helping the catalytic center reengage with nascent RNA 3´-terminus during abortive synthesis. The chapter describes four new methods useful for biochemical and structure-functional studies of Tth Gre factors: direct chromatographic assay for competitive binding of GreA1 and GreA2 to RNAP, specific transcript cleavage (misincorporation–excision) assay for GreA1, inhibition of RNA synthesis assay for GreA2, and localized Fe2 + -induced hydroxyl radical mapping of GreA1 and GreA2 sites proximal to RNAP catalytic center.

Early Transcriptional Arrest at Escherichia coli rplN and ompX Promoters

Bacterial transcription elongation factors GreA and GreB stimulate the intrinsic RNase activity of RNA polymerase (RNAP), thus helping the enzyme to read through pausing and arresting sites on DNA. Gre factors also accelerate RNAP transition from initiation to elongation. Here, we characterized the molecular mechanism by which Gre factors facilitate transcription at two Escherichia coli promoters, PrplN and PompX, that require GreA for optimal in vivo activity. Using in vitro transcription assays, KMnO4 footprinting, and Fe2+-induced hydroxyl radical mapping, we show that during transcription initiation at PrplN and PompX in the absence of Gre factors, RNAP falls into a condition of promoter-proximal transcriptional arrest that prevents production of full-length transcripts both in vitro and in vivo. Arrest occurs when RNAP synthesizes 9–14-nucleotide-long transcripts and backtracks by 5–7 (PrplN) or 2–4 (PompX) nucleotides. Initiation factor σ70 contributes to the formation of arrested complexes at both promoters. The signal for promoter-proximal arrest at PrplN is bipartite and requires two elements: the extended −10 promoter element and the initial transcribed region from positions +2 to +6. GreA and GreB prevent arrest at PrplN and PompX by inducing cleavage of the 3′-proximal backtracked portion of RNA at the onset of arrested complex formation and stimulate productive transcription by allowing RNAP to elongate the 5′-proximal transcript cleavage products in the presence of substrates. We propose that promoter-proximal arrest is a common feature of many bacterial promoters and may represent an important physiological target of regulation by transcript cleavage factors.

Early transcriptional arrest at E. coli rplN and ompX promoter

Bacterial transcription elongation factors GreA and GreB stimulate the intrinsic RNase activity of RNA polymerase (RNAP), thus helping the enzyme to read through pausing and arresting sites on DNA. Gre factors also accelerate RNAP transition from initiation to elongation. Here, we characterized the molecular mechanism by which Gre factors facilitate transcription at two Escherichia coli promoters, PrplN and PompX, that require GreA for optimal in vivo activity. Using in vitro transcription assays, KMnO4 footprinting, and Fe2+-induced hydroxyl radical mapping, we show that during transcription initiation at PrplN and PompX in the absence of Gre factors, RNAP falls into a condition of promoter-proximal transcriptional arrest that prevents production of full-length transcripts both in vitro and in vivo. Arrest occurs when RNAP synthesizes 9–14-nucleotide-long transcripts and backtracks by 5–7 (PrplN) or 2–4 (PompX) nucleotides. Initiation factor σ70 contributes to the formation of arrested complexes at both promoters. The signal for promoter-proximal arrest at PrplN is bipartite and requires two elements: the extended −10 promoter element and the initial transcribed region from positions +2 to +6. GreA and GreB prevent arrest at PrplN and PompX by inducing cleavage of the 3′-proximal backtracked portion of RNA at the onset of arrested complex formation and stimulate productive transcription by allowing RNAP to elongate the 5′-proximal transcript cleavage products in the presence of substrates. We propose that promoter-proximal arrest is a common feature of many bacterial promoters and may represent an important physiological target of regulation by transcript cleavage factors.