Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase.

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis.

We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in Bacillus subtilis YB955 (hisC952 metB5 leuC427). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed B. subtilis YB955 cells deficient in the GO or AP endonuclease repair proteins. To this end, mfd and greA were disabled in genetic backgrounds defective in the GO and AP endonuclease repair proteins, and the strains were tested for growth-associated and stress-associated mutagenesis. The results revealed that disruption of mfd or greA abrogated the production of stress-associated amino acid revertants in the GO and nfo exoA nth strains, respectively. These results suggest that in nutritionally stressed B. subtilis cells, spontaneous nonbulky DNA lesions are processed in an error-prone manner with the participation of Mfd and GreA. In support of this notion, stationary-phase ΔytkD ΔmutM ΔmutY (referred to here as ΔGO) and Δnfo ΔexoA Δnth (referred to here as ΔAP) cells accumulated 8-oxoguanine (8-OxoG) lesions, which increased significantly following Mfd disruption. In contrast, during exponential growth, disruption of mfd or greA increased the production of His+, Met+, or Leu+ prototrophs in both DNA repair-deficient strains. Thus, in addition to unveiling a role for GreA in mutagenesis, our results suggest that Mfd and GreA promote or prevent mutagenic events driven by spontaneous genetic lesions during the life cycle of B. subtilisIMPORTANCE In this paper, we report that spontaneous genetic lesions of an oxidative nature in growing and nutritionally stressed B. subtilis strain YB955 (hisC952 metB5 leuC427) cells drive Mfd- and GreA-dependent repair transactions. However, whereas Mfd and GreA elicit faithful repair events during growth to maintain genome fidelity, under starving conditions, both factors promote error-prone repair to produce genetic diversity, allowing B. subtilis to escape from growth-limiting conditions.

Interactions in the active site of Deinococcus radiodurans RNA polymerase during RNA proofreading.

Co-transcriptional RNA proofreading by RNA polymerase (RNAP) is essential for accurate mRNA synthesis and reactivation of stalled transcription complexes, which can otherwise compromise genome integrity. RNAP from the stress-resistant bacterium Deinococcus radiodurans exhibits high levels of RNA cleavage in comparison with RNAP from Escherichia coli, which allows it to remove misincorporated nucleotides with high efficiency. Here, we show that the rate of RNA cleavage by D. radiodurans RNAP depends on the structure of the (mis)matched RNA 3'-nucleotide and its contacts with the active site. These interactions likely position the reactive phosphodiester bond in the cleavage-competent conformation, thus facilitating its hydrolysis catalyzed by metal ions in the active center. The universal RNA cleavage factor GreA largely alleviates defects in RNA cleavage caused by modifications in the RNA 3'-nucleotide or in its binding pocket in RNAP, suggesting that GreA functionally substitutes for these contacts. The results demonstrate that various RNAPs rely on a conserved mechanism for RNA proofreading, which can be modulated by changes in accessory parts of the active center.

Autoregulation of greA Expression Relies on GraL Rather than on greA Promoter Region.

GreA is a well-characterized transcriptional factor that acts primarily by rescuing stalled RNA polymerase complexes, but has also been shown to be the major transcriptional fidelity and proofreading factor, while it inhibits DNA break repair. Regulation of greA gene expression itself is still not well understood. So far, it has been shown that its expression is driven by two overlapping promoters and that greA leader encodes a small RNA (GraL) that is acting in trans on nudE mRNA. It has been also shown that GreA autoinhibits its own expression in vivo. Here, we decided to investigate the inner workings of this autoregulatory loop. Transcriptional fusions with lacZ reporter carrying different modifications (made both to the greA promoter and leader regions) were made to pinpoint the sequences responsible for this autoregulation, while GraL levels were also monitored. Our data indicate that GreA mediated regulation of its own gene expression is dependent on GraL acting in cis (a rare example of dual-action sRNA), rather than on the promoter region. However, a yet unidentified, additional factor seems to participate in this regulation as well. Overall, the GreA/GraL regulatory loop seems to have unique but hard to classify properties.

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases.

RNA cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA cleavage. RNA cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli, Deinococcus radiodurans, and Thermus aquaticus, we show that the functions of the TL and Gre factors in RNA cleavage are conserved in various species, with important variations that may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA cleavage by all three RNAPs and eliminate the interspecies differences in the reaction rates. GreA factors activate RNA cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent cleavage are lower for ΔTL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA cleavage in certain types of backtracked transcription complexes, suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation.

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.

Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness.

There is growing evidence that GreA aids adaptation to stressful environments in various bacteria. However, the functions of GreA among mycobacteria remain obscure. Here, we report on cellular consequences following deletion of greA gene in Mycobacterium spp. The greA mutant strain (ΔgreA) was generated in Mycobacterium smegmatis, Mycobacterium tuberculosis (MTB) H37Ra, and M. tuberculosis H37Rv. Deletion of greA results in growth retardation and poor survival in response to adverse stress, besides rendering M. tuberculosis more susceptible to vancomycin and rifampicin. By using RNA-seq, we observe that disrupting greA results in the differential regulation of 195 genes in M. smegmatis with 167 being negatively regulated. Among these, KEGG pathways significantly enriched for differentially regulated genes included tryptophan metabolism, starch and sucrose metabolism, and carotenoid biosynthesis, supporting a role of GreA in the metabolic regulation of mycobacteria. Moreover, like Escherichia coli GreA, M. smegmatis GreA exhibits a series of conservative features, and the anti-backtracking activity of C-terminal domain is indispensable for the expression of glgX, a gene was down-regulated in the RNA-seq data. Interestingly, the decrease in the expression of glgX by CRISPR interference, resulted in reduced growth. Finally, intracellular fitness significantly declines due to loss of greA. Our data indicates that GreA is an important factor for the survival and resistance establishment in Mycobacterium spp. This study provides new insight into GreA as a potential target in multi-drug resistant TB treatment.

Transcription fidelity: New paradigms in epigenetic inheritance, genome instability and disease.

RNA transcription errors are transient, yet frequent, events that do have consequences for the cell. However, until recently we lacked the tools to empirically measure and study these errors. Advances in RNA library preparation and next generation sequencing (NGS) have allowed the spectrum of transcription errors to be empirically measured over the entire transcriptome and in nascent transcripts. Combining these powerful methods with forward and reverse genetic strategies has refined our understanding of transcription factors known to enhance RNA accuracy and will enable the discovery of new candidates. Furthermore, these approaches will shed additional light on the complex interplay between transcription fidelity and other DNA transactions, such as replication and repair, and explore a role for transcription errors in cellular evolution and disease.

A search for the in trans role of GraL, an Escherichia coli small RNA.

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL's targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target's activity (nudE) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA, the nudE::lacZ fusion's ß-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA. Furthermore, overproduction of GreA alone increased the nudE::lacZ fusion's activity as well. This suggests existence of complex regulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we performed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL-nudE complexes were not detected, even though the detectable amount of unbound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it. We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are rendered essential by GraL deletion, however, they are involved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraL's mechanism of action is non-canonical, unique and requires further research.

A Genome-Wide Assay Specifies Only GreA as a Transcription Fidelity Factor in Escherichia coli.

Although mutations are the basis for adaptation and heritable genetic change, transient errors occur during transcription at rates that are orders of magnitude higher than the mutation rate. High rates of transcription errors can be detrimental by causing the production of erroneous proteins that need to be degraded. Two transcription fidelity factors, GreA and GreB, have previously been reported to stimulate the removal of errors that occur during transcription, and a third fidelity factor, DksA, is thought to decrease the error rate through an unknown mechanism. Because the majority of transcription-error assays of these fidelity factors were performed in vitro and on individual genes, we measured the in vivo transcriptome-wide error rates in all possible combinations of mutants of the three fidelity factors. This method expands measurements of these fidelity factors to the full spectrum of errors across the entire genome. Our assay shows that GreB and DksA have no significant effect on transcription error rates, and that GreA only influences the transcription error rate by reducing G-to-A errors.