Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism.

The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.

Obstacles to Scanning by RNase E Govern Bacterial mRNA Lifetimes by Hindering Access to Distal Cleavage Sites.

The diversity of mRNA lifetimes in bacterial cells is difficult to reconcile with the relaxed cleavage site specificity of RNase E, the endonuclease most important for governing mRNA degradation. This enzyme has generally been thought to locate cleavage sites by searching freely in three dimensions. However, our results now show that its access to such sites in 5'-monophosphorylated RNA is hindered by obstacles-such as bound proteins or ribosomes or coaxial small RNA (sRNA) base pairing-that disrupt the path from the 5' end to those sites and prolong mRNA lifetimes. These findings suggest that RNase E searches for cleavage sites by scanning linearly from the 5'-terminal monophosphate along single-stranded regions of RNA and that its progress is impeded by structural discontinuities encountered along the way. This discovery has major implications for gene regulation in bacteria and suggests a general mechanism by which other prokaryotic and eukaryotic regulatory proteins can be controlled.

NYEs/SGRs-mediated chlorophyll degradation is critical for detoxification during seed maturation in Arabidopsis.

In the seed industry, chlorophyll (Chl) fluorescence is often used as a major non-invasive reporter of seed maturation and quality. Breakdown of Chl is a proactive process during the late stage of seed maturation, as well as during leaf senescence and fruit ripening. However, the biological significance of this process is still unclear. NYE1 and NYE2 are Mg-dechelatases, catalyzing the first rate-limiting step of Chl a degradation. Loss-of-function of both NYE1 and NYE2 not only results in a nearly complete retention of Chl during leaf senescence, but also produces green seeds in Arabidopsis. In this study, we showed that Chl retention in the nye1 nye2 double-mutant caused severe photo-damage to maturing seeds. Upon prolonged light exposure, green seeds of nye1 nye2 gradually bleached out and eventually lost their germination capacity. This organ-specific photosensitive phenotype is likely due to an over-accumulation of free Chl, which possesses photosensitizing properties and causes a burst of reactive oxygen species upon light exposure. As expected, a similar, albeit much milder, photosensitive phenotype was observed in the seeds of d1 d2, a green-seed mutant defective in NYE/SGR orthologous genes in soybean. Taken together, our data suggest that efficient NYEs-mediated Chl degradation is critical for detoxification during seed maturation.

SgrT, a Small Protein That Packs a Sweet Punch.

The SgrS small RNA (sRNA) has been shown to protect against elevated levels of glucose phosphate by regulating the stability and translation of mRNAs encoding proteins involved in sugar transport and catabolism. The sRNA also was known to encode a protective 43-amino-acid protein, SgrT, but little was known about its mechanism of action. Lloyd et al. (J Bacteriol 199:e00869-16, 2017, https://doi.org/10.1128/JB.00869-16) use cell biological and genetic approaches to demonstrate that the small protein interacts with the PtsG importer to block glucose transport by this phosphotransferase system and promote utilization of nonpreferred carbon sources to maintain growth during glucose-phosphate stress.

Stringent Response Regulators Contribute to Recovery from Glucose Phosphate Stress in Escherichia coli.

In enteric bacteria such as Escherichia coli, the transcription factor SgrR and the small RNA SgrS regulate the response to glucose phosphate stress, a metabolic dysfunction that results in growth inhibition and stems from the intracellular accumulation of sugar phosphates. SgrR activates the transcription of sgrS, and SgrS helps to rescue cells from stress in part by inhibiting the uptake of stressor sugar phosphates. While the regulatory targets of this stress response are well described, less is known about how the SgrR-SgrS response itself is regulated. To further characterize the regulation of the glucose phosphate stress response, we screened global regulator gene mutants for growth changes during glucose phosphate stress. We found that deleting dksA, which encodes a regulator of the stringent response to nutrient starvation, decreases growth under glucose phosphate stress conditions. The stringent response alarmone regulator ppGpp (synthesized by RelA and SpoT) also contributes to recovery from glucose phosphate stress: as with dksA, mutating relA and spoT worsens the growth defect of an sgrS mutant during stress, although the sgrS relA spoT mutant defect was only detectable under lower stress levels. In addition, mutating dksA or relA and spoT lowers sgrS expression (as measured with a P sgrS -lacZ fusion), suggesting that the observed growth defects may be due to decreased induction of the glucose phosphate stress response or related targets. This regulatory effect could occur through altered sgrR transcription, as dksA and relA spoT mutants also exhibit decreased expression of a P sgrR -lacZ fusion. Taken together, this work supports a role for stringent response regulators in aiding the recovery from glucose phosphate stress.IMPORTANCE Glucose phosphate stress leads to growth inhibition in bacteria such as Escherichia coli when certain sugar phosphates accumulate in the cell. The transcription factor SgrR and the small RNA SgrS alleviate this stress in part by preventing further sugar phosphate transport. While the regulatory mechanisms of this response have been characterized, the regulation of the SgrR-SgrS response itself is not as well understood. Here, we describe a role for stringent response regulators DksA and ppGpp in the response to glucose phosphate stress. sgrS dksA and sgrS relA spoT mutants exhibit growth defects under glucose phosphate stress conditions. These defects may be due to a decrease in stress response induction, as deleting dksA or relA and spoT also results in decreased expression of sgrS and sgrR This research presents one of the first regulatory effects on the glucose phosphate stress response outside SgrR and SgrS and depicts a novel connection between these two metabolic stress responses.

New developments in post-transcriptional regulation of operons by small RNAs.

Small regulatory RNAs (sRNAs) are influential post-transcriptional modulators of gene expression in bacteria. They regulate gene expression by base pairing to target mRNAs, leading to inhibition of translation and/or alteration of mRNA stability. Recently, several sRNAs have been discovered to regulate genes encoded in operons. In some cases, these sRNAs regulate all the genes encoded by the polycistronic mRNA (coordinate regulation) while in other cases, only a select subset of cistrons is controlled by the sRNA (discoordinate regulation). In this point of view, mechanisms of regulation and characteristics of sRNA-mRNA interactions involving polycistronic mRNAs are described. The diversity in mechanisms represented by these few characterized examples suggests that we still have much to learn about sRNA regulation of long polycistronic messages.

Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli.

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

Regulation of Transcription Termination of Small RNAs and by Small RNAs: Molecular Mechanisms and Biological Functions.

Accurate and efficient transcription termination is an important step for cells to generate functional RNA transcripts. In bacteria, two mechanisms are responsible for terminating transcription: intrinsic (Rho-independent) termination and Rho-dependent termination. Growing examples suggest that neither type of transcription termination is static, but instead are highly dynamic and regulated. Regulatory small RNAs (sRNAs) are key players in bacterial stress responses, are frequently expressed under specific growth conditions, and are predominantly terminated through the intrinsic termination mechanism. Once made, sRNAs can base-pair with mRNA targets and regulate mRNA translation and stability. Recent findings suggest that alterations in the efficiency of intrinsic termination for sRNAs under various growth conditions may affect the availability of a given sRNA and the ability of the sRNA to function properly. Moreover, alterations of mRNA structure, translation, and accessibility by sRNAs have the potential to impact the access of Rho factor to mRNAs and thus termination of the mRNA. Indeed, recent studies have revealed that some sRNAs can modulate target gene expression by stimulating or inhibiting Rho-dependent termination, thus expanding the regulatory power of bacterial sRNAs. Here we review the current knowledge on intrinsic termination of sRNAs and sRNA-mediated Rho-dependent termination of protein coding genes in bacteria.

Functional and RNA-Sequencing Analysis Revealed Expression of a Novel Stay-Green Gene from Zoysia japonica (ZjSGR) Caused Chlorophyll Degradation and Accelerated Senescence in Arabidopsis.

Senescence is not only an important developmental process, but also a responsive regulation to abiotic and biotic stress for plants. Stay-green protein plays crucial roles in plant senescence and chlorophyll degradation. However, the underlying mechanisms were not well-studied, particularly in non-model plants. In this study, a novel stay-green gene, ZjSGR, was isolated from Zoysia japonica. Subcellular localization result demonstrated that ZjSGR was localized in the chloroplasts. Quantitative real-time PCR results together with promoter activity determination using transgenic Arabidopsis confirmed that ZjSGR could be induced by darkness, ABA and MeJA. Its expression levels could also be up-regulated by natural senescence, but suppressed by SA treatments. Overexpression of ZjSGR in Arabidopsis resulted in a rapid yellowing phenotype; complementary experiments proved that ZjSGR was a functional homolog of AtNYE1 from Arabidopsis thaliana. Over expression of ZjSGR accelerated chlorophyll degradation and impaired photosynthesis in Arabidopsis. Transmission electron microscopy observation revealed that overexpression of ZjSGR decomposed the chloroplasts structure. RNA sequencing analysis showed that ZjSGR could play multiple roles in senescence and chlorophyll degradation by regulating hormone signal transduction and the expression of a large number of senescence and environmental stress related genes. Our study provides a better understanding of the roles of SGRs, and new insight into the senescence and chlorophyll degradation mechanisms in plants.

Tn5 transposition in Escherichia coli is repressed by Hfq and activated by over-expression of the small non-coding RNA SgrS.

BACKGROUND: Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base pairing of mRNAs with trans-encoded sRNAs. It was previously shown that Hfq down-regulates Tn10 transposition by inhibiting IS10 transposase expression at the post-transcriptional level. This provided the first example of Hfq playing a role in DNA transposition and led us to ask if a related transposon, Tn5, is similarly regulated. RESULTS: We show that Hfq strongly suppresses Tn5 transposition in Escherichia coli by inhibiting IS50 transposase expression. However, in contrast to the situation for Tn10, Hfq primarily inhibits IS50 transposase transcription. As Hfq does not typically function directly in transcription, we searched for a transcription factor that also down-regulated IS50 transposase transcription and is itself under Hfq control. We show that Crp (cyclic AMP receptor protein) fits these criteria as: (1) disruption of the crp gene led to an increase in IS50 transposase expression and the magnitude of this increase was comparable to that observed for an hfq disruption; and (2) Crp expression decreased in hfq (-) . We also demonstrate that IS50 transposase expression and Tn5 transposition are induced by over-expression of the sRNA SgrS and link this response to glucose limitation. CONCLUSIONS: Tn5 transposition is negatively regulated by Hfq primarily through inhibition of IS50 transposase transcription. Preliminary results support the possibility that this regulation is mediated through Crp. We also provide evidence that glucose limitation activates IS50 transposase transcription and transposition.