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.
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.
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.
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 σ subunit of 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.
The σ subunit of bacterial RNA polymerase is required for promoter recognition during transcription initiation but may also regulate transcription elongation. The principal σ 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 σ 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.
Besides X-family DNA polymerases (first of all, Pol β) several other human DNA polymerases from Yand 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.Human DNA polymerase iota (Pol ι) belongs to the Y-family of translesion DNA polymerases and demonstrates very low accuracy of DNA synthesis. The high error rate of Pol ι is a result of the special organization of the active site 1, 2 which is adapted to bypass a variety of DNA lesions 3 , including bulky carcinogenic lesions 4-9 and interstrand DNA cross-links 10 . The DNA polymerase activity of Pol ι is stimulated by Mn 2+ ions 11,12 . In addition to the DNA polymerase activity, human Pol ι has an intrinsic 5′-deoxyribose-5-phosphate lyase activity (dRP-lyase activity) 13,14 . Pol ι carries out efficient DNA synthesis on gapped DNA substrates and in reactions reconstituted with uracil-DNA glycosylase, AP-endonuclease and ligase can repair DNA 13,15 . Moreover, Pol ι is able to complement in vitro the short-patch base excision repair (BER) deficiency of Pol β null cell extracts 16 .
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.
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