Key interactions of RNA polymerase with 6S RNA and secondary channel factors during pRNA synthesis.

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.

Highly specific aptamer trap for extremophilic RNA polymerases.

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus phylum. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.

Phenotypic Test of Benzo[4,5]imidazo[1,2-c]pyrimidinone-Based Nucleoside and Non-Nucleoside Derivatives against DNA and RNA Viruses, Including Coronaviruses.

Emerging and re-emerging viruses periodically cause outbreaks and epidemics around the world, which ultimately lead to global events such as the COVID-19 pandemic. Thus, the urgent need for new antiviral drugs is obvious. Over more than a century of antiviral development, nucleoside analogs have proven to be promising agents against diversified DNA and RNA viruses. Here, we present the synthesis and evaluation of the antiviral activity of nucleoside analogs and their deglycosylated derivatives based on a hydroxybenzo[4,5]imidazo[1,2-c]pyrimidin-1(2H)-one scaffold. The antiviral activity was evaluated against a panel of structurally and phylogenetically diverse RNA and DNA viruses. The leader compound showed micromolar activity against representatives of the family Coronaviridae, including SARS-CoV-2, as well as against respiratory syncytial virus in a submicromolar range without noticeable toxicity for the host cells. Surprisingly, methylation of the aromatic hydroxyl group of the leader compound resulted in micromolar activity against the varicella-zoster virus without any significant impact on cell viability. The leader compound was shown to be a weak inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase. It also inhibited biocondensate formation important for SARS-CoV-2 replication. The active compounds may be considered as a good starting point for further structure optimization and mechanistic and preclinical studies.

Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA.

The SARS-CoV-2 betacoronavirus pandemic has claimed more than 6.5 million lives and, despite the development and use of COVID-19 vaccines, remains a major global public health problem. The development of specific drugs for the treatment of this disease remains a very urgent task. In the context of a repurposing strategy, we previously screened a library of nucleoside analogs showing different types of biological activity against the SARS-CoV-2 virus. The screening revealed compounds capable of inhibiting the reproduction of SARS-CoV-2 with EC50 values in the range of 20-50 µM. Here we present the design and synthesis of various analogs of the leader compounds, the evaluation of their cytotoxicity and antiviral activity against SARS-CoV-2 in cell cultures, as well as experimental data on RNA-dependent RNA polymerase inhibition. Several compounds have been shown to prevent the interaction between the SARS-CoV-2 RNA-dependent RNA polymerase and the RNA substrate, likely inhibiting virus replication. Three of the synthesized compounds have also been shown to inhibit influenza virus. The structures of these compounds can be used for further optimization in order to develop an antiviral drug.

Effects of natural polymorphisms in SARS-CoV-2 RNA-dependent RNA polymerase on its activity and sensitivity to inhibitors in vitro.

SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is the key enzyme required for viral replication and mRNA synthesis. RdRp is one of the most conserved viral proteins and a promising target for antiviral drugs and inhibitors. At the same time, analysis of public databases reveals multiple variants of SARS-CoV-2 genomes with substitutions in the catalytic RdRp subunit nsp12. Structural mapping of these mutations suggests that some of them may affect the interactions of nsp12 with its cofactors nsp7/nsp8 as well as with RNA substrates. We have obtained several mutations of these types and demonstrated that some of them decrease specific activity of RdRp in vitro, possibly by changing RdRp assembly and/or its interactions with RNA. Therefore, natural polymorphisms in RdRp may potentially affect viral replication. Furthermore, we have synthesized a series of polyphenol and diketoacid derivatives based on previously studied inhibitors of hepatitis C virus RdRp and found that several of them can inhibit SARS-CoV-2 RdRp. Tested mutations in RdRp do not have strong effects on the efficiency of inhibition. Further development of more efficient non-nucleoside inhibitors of SARS-CoV-2 RdRp should take into account the existence of multiple polymorphic variants of RdRp.

Effects of natural RNA modifications on the activity of SARS-CoV-2 RNA-dependent RNA polymerase.

RNA-dependent RNA polymerase (RdRp) plays a key role in the replication of RNA viruses, including SARS-CoV-2. Processive RNA synthesis by RdRp is crucial for successful genome replication and expression, especially in the case of very long coronaviral genomes. Here, we analysed the activity of SARS-CoV-2 RdRp (the nsp12-nsp7-nsp8 complex) on synthetic primer-templates of various structures, including substrates with mismatched primers or template RNA modifications. It has been shown that RdRp cannot efficiently extend RNA primers containing mismatches and has no intrinsic RNA cleavage activity to remove the primer 3'-end, thus necessitating the action of exoribonuclease for proofreading. Similar to DNA-dependent RNA polymerases, RdRp can perform processive pyrophosphorolysis of the nascent RNA product but this reaction is also blocked in the presence of mismatches. Furthermore, we have demonstrated that several natural post-transcriptional modifications in the RNA template, which do not prevent complementary interactions (N6-methyladenosine, 5-methylcytosine, inosine and pseudouridine), do not change RdRp processivity. At the same time, certain modifications of RNA bases and ribose residues strongly block RNA synthesis, either prior to nucleotide incorporation (3-methyluridine and 1-methylguanosine) or immediately after it (2'-O-methylation). The results demonstrate that the activity of SARS-CoV-2 RdRp can be strongly inhibited by common modifications of the RNA template suggesting a way to design novel antiviral compounds.

Suppressor mutations in Escherichia coli RNA polymerase alter transcription initiation but do not affect translesion RNA synthesis in vitro.

Bacterial RNA polymerase (RNAP) coordinates transcription with DNA repair and replication. Many RNAP mutations have pleiotropic phenotypes with profound effects on transcription-coupled processes. One class of RNAP mutations (rpo∗) has been shown to suppress mutations in regulatory factors responsible for changes in gene expression during stationary phase or starvation, as well as in factors involved in the restoration of replication forks after DNA damage. These mutations were suggested to affect the ability of RNAP to transcribe damaged DNA and to decrease the stability of transcription complexes, thus facilitating their dislodging during DNA replication and repair, although this was not explicitly demonstrated. Here, we obtained nine mutations of this class located around the DNA/RNA binding cleft of Escherichia coli RNAP and analyzed their transcription properties in vitro. We found that these mutations decreased promoter complex stability to varying degrees, and all decreased the activity of rRNA promoters. However, they did not have strong effects on elongation complex stability. Some mutations were shown to stimulate transcriptional pauses or decrease intrinsic RNA cleavage by RNAP, but none altered the ability of RNAP to transcribe DNA templates containing damaged nucleotides. Thus, we conclude that the suppressor phenotypes of the mutations are unlikely to result from direct effects on DNA lesion recognition by RNAP but may be primarily explained by changes in transcription initiation. Further analysis of the effects of these mutations on the genomic distribution of RNAP and its interactions with regulatory factors will be essential for understanding their diverse phenotypes in vivo.

Universal functions of the σ finger in alternative σ factors during transcription initiation by bacterial RNA polymerase.

The bacterial σ factor plays the central role in promoter recognition by RNA polymerase (RNAP). The primary σ factor, involved in transcription of housekeeping genes, was also shown to participate in the initiation of RNA synthesis and promoter escape by RNAP. In the open promoter complex, the σ finger formed by σ region 3.2 directly interacts with the template DNA strand upstream of the transcription start site. Here, we analysed the role of the σ finger in transcription initiation by four alternative σ factors in Escherichia coli, σ38, σ32, σ28 and σ24. We found that deletions of the σ finger to various extent compromise the activity of RNAP holoenzymes containing alternative σ factors, especially at low NTP concentrations. All four σs are able to utilize NADH as a noncanonical priming substrate but it has only mild effects on the efficiency of transcription initiation. The mediators of the stringent response, transcription factor DksA and the alarmone ppGpp decrease RNAP activity and promoter complex stability for all four σ factors on tested promoters. For all σs except σ38, deletions of the σ finger conversely increase the stability of promoter complexes and decrease their sensitivity to DksA and ppGpp. The result suggests that the σ finger plays a universal role in transcription initiation by alternative σ factors and sensitizes promoter complexes to the action of global transcription regulators DksA and ppGpp by modulating promoter complex stability.

Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein.

In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.

Rewiring of growth-dependent transcription regulation by a point mutation in region 1.1 of the housekeeping σ factor.

In bacteria, rapid adaptation to changing environmental conditions depends on the interplay between housekeeping and alternative σ factors, responsible for transcription of specific regulons by RNA polymerase (RNAP). In comparison with alternative σ factors, primary σs contain poorly conserved region 1.1, whose functions in transcription are only partially understood. We found that a single mutation in region 1.1 in Escherichia coli σ70 rewires transcription regulation during cell growth resulting in profound phenotypic changes. Despite its destabilizing effect on promoter complexes, this mutation increases the activity of rRNA promoters and also decreases RNAP sensitivity to the major regulator of stringent response DksA. Using total RNA sequencing combined with single-cell analysis of gene expression we showed that changes in region 1.1 disrupt the balance between the "greed" and "fear" strategies thus making the cells more susceptible to environmental threats and antibiotics. Our results reveal an unexpected role of σ region 1.1 in growth-dependent transcription regulation and suggest that changes in this region may facilitate rapid switching of RNAP properties in evolving bacterial populations.

Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA.

The σ factor drives promoter recognition by bacterial RNA polymerase (RNAP) and is also essential for later steps of transcription initiation, including RNA priming and promoter escape. Conserved region 3.2 of the primary σ factor ('σ finger') directly contacts the template DNA strand in the open promoter complex and facilitates initiating NTP binding in the active center of RNAP. Ribosomal RNA promoters are responsible for most RNA synthesis during exponential growth but should be silenced during the stationary phase to save cell resources. In Escherichia coli, the silencing mainly results from the action of the secondary channel factor DksA, which together with ppGpp binds RNAP and dramatically decreases the stability of intrinsically unstable rRNA promoter complexes. We demonstrate that this switch depends on the σ finger that destabilizes RNAP-promoter interactions. Mutations in the σ finger moderately decrease initiating NTP binding but significantly increase promoter complex stability and reduce DksA affinity to the RNAP-rRNA promoter complex, thus making rRNA transcription less sensitive to DksA/ppGpp both in vitro and in vivo. Thus, destabilization of rRNA promoter complexes by the σ finger makes them a target for robust regulation by the stringent response factors under stress conditions.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria.

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Interplay between σ region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase.

In bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5'-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ70 subunit of Escherichia coli RNAP. We show that substitutions and deletions in σ region 3.2 decrease the rate of promoter escape and lead to accumulation of inactive complexes during transcription initiation. Secondary channel factors differentially regulate this process depending on the promoter and mutations in σ region 3.2. GreA generally increase the rate of promoter escape; DksA also stimulates promoter escape on certain templates, while GreB either stimulates or inhibits this process depending on the template. When observed, the stimulation of promoter escape correlates with the accumulation of stressed transcription complexes with scrunched DNA, while changes in the RNA 5'-end structure modulate promoter clearance. Thus, the initiation-to-elongation transition is controlled by a complex interplay between RNAP-binding protein factors and the growing RNA chain.

Identification of amino acid residues involved in the dRP-lyase activity of human Pol ι.

Besides X-family DNA polymerases (first of all, Pol ß) several other human DNA polymerases from Y- and A- families were shown to possess the dRP-lyase activity and could serve as backup polymerases in base excision repair (Pol ι, Rev1, Pol γ and Pol θ). However the exact position of the active sites and the amino acid residues involved in the dRP-lyase activity in Y- and A- family DNA polymerases are not known. Here we carried out functional analysis of fifteen amino acid residues possibly involved in the dRP-lyase activity of human Pol ι. We show that substitutions of residues Q59, K60 and K207 impair the dRP-lyase activity of Pol ι while residues in the HhH motif of the thumb domain are dispensable for this activity. While both K60G and K207A substitutions decrease Schiff-base intermediate formation during dRP group cleavage, the latter substitution also strongly affects the DNA polymerase activity of Pol ι, suggesting that it may impair DNA binding. These data are consistent with an important role of the N-terminal region in the dRP-lyase activity of Pol ι, with possible involvement of residues from the finger domain in the dRP group cleavage.

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

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.

σ38-dependent promoter-proximal pausing by bacterial RNA polymerase.

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.

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.