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Rat1 is a 5′→3′ exoribonuclease in budding yeast. It is a highly conserved protein with homologs being present in fission yeast, flies, worms, mice and humans. Rat1 and its human homolog Xrn2 have been implicated in multiple nuclear processes. Here we report a novel role of Rat1 in mRNA splicing. We observed an increase in the level of unspliced transcripts in mutants of Rat1. Accumulation of unspliced transcripts was not due to the surveillance role of Rat1 in degrading unspliced mRNA, or an indirect effect of Rat1 function in termination of transcription or on the level of splicing factors in the cell, or due to an increased elongation rate in Rat1 mutants. ChIP-Seq analysis revealed Rat1 crosslinking to the introns of a subset of yeast genes. Mass spectrometry and coimmunoprecipitation revealed an interaction of Rat1 with the Clf1, Isy1, Yju2, Prp43 and Sub2 splicing factors. Furthermore, recruitment of splicing factors on the intron was compromised in the Rat1 mutant. Based on these findings we propose that Rat1 has a novel role in splicing of mRNA in budding yeast. Rat1, however, is not a general splicing factor as it crosslinks to only long introns with an average length of 400 nucleotides.

Transcription in cyanobacteria involves several fascinating features. Cyanobacteria comprise one of the very few groups in which no proofreading factors (Gre homologues) have been identified. Gre factors increase the efficiency of RNA cleavage, therefore helping to maintain the fidelity of the RNA transcript and assist in the resolution of stalled RNAPs to prevent genome damage. The vast majority of bacterial species encode at least one of these highly conserved factors and so their absence in cyanobacteria is intriguing. Additionally, the largest subunit of bacterial RNAP has undergone a split in cyanobacteria to form two subunits and the SI3 insertion within the integral trigger loop element is roughly 3.5 times larger than in Escherichia coli. The Rho termination factor also appears to be absent, leaving cyanobacteria to rely solely on an intrinsic termination mechanism. Furthermore, cyanobacteria must be able to respond to environment signals such as light intensity and tightly synchronise gene expression and other cell activities to a circadian rhythm.

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/β domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination., Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2014-53762-P to B.G., BFU2015-71978-REDT and BFU2013-48374-P to O.C., CTQ2011-26665 and CTQ2014-52633 to J.M.P.C.]; E.F.-E. was supported by BFU2014–53762-P grant. Funding for open access charge: MINECO.

Summary CPSF100 is a core component of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex for the 3′-end formation of mRNA, but it still has no clear functional assignment. CPSF100 was reported to play a role in RNA silencing and promote flowering in Arabidopsis. However, the molecular mechanisms underlying these phenomena are not fully understood. Our genetics analyses indicate that plants with a hypomorphic mutant of CPSF100 (esp5) show defects in embryogenesis, reduced seed production, or altered root morphology. To unravel this puzzle, we employed a poly(A) tag sequencing (PAT-seq) protocol and uncovered a different poly(A) profile in esp5. This transcriptome-wide analyses revealed alternative polyadenylation (APA) of thousands of genes, most of which result in transcriptional read-through in protein-coding genes. AtCPSF100 also affects poly(A) signal recognition on the far-upstream elements, particularly it prefers less U-rich sequences. Importantly, AtCPSF100 was found to exert its functions through the change of poly(A) sites on genes encoding binding proteins, such as nucleotide binding, RNA binding and poly(U) binding proteins. In addition, through its interaction with RNA Polymerase II C-terminal domain (CTD) and affecting the expression level of CTD phosphatase-like 3 (CPL3), AtCPSF100 is shown to potentially ensure transcriptional termination by dephosphorylation of Ser2 on the CTD. These data suggest a key role of CPSF100 in locating poly(A) sites and impacting transcription termination. This article is protected by copyright. All rights reserved.

There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2'-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2'-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.

If not repaired, ultraviolet light-induced DNA damage can lead to genome instability. Nucleotide excision repair (NER) of UV photoproducts is generally fast in the coding region of genes, where RNA polymerase-II (RNAP2) arrest at damage sites and trigger transcription-coupled NER (TC-NER). In Saccharomyces cerevisiae, there is RNA polymerase-I (RNAP1)-dependent TC-NER, but this process remains elusive. Therefore, we wished to characterize TC-NER efficiency in different regions of the rDNA locus: where RNAP1 are present at high density and start transcription elongation, where the elongation rate is slow, and in the transcription terminator where RNAP1 pause, accumulate and then are released. The Rpa12 subunit of RNAP1 and the Nsi1 protein participate in transcription termination, and NER efficiency was compared between wild type and cells lacking Rpa12 or Nsi1. The presence of RNAP1 was determined by chromatin endogenous cleavage and chromatin immunoprecipitation, and repair was followed at nucleotide precision with an assay that is based on the blockage of Taq polymerase by UV photoproducts. We describe that TC-NER, which is modulated by the RNAP1 level and elongation rate, ends at the 35S rRNA gene transcription termination site.

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome., Author summary Many cellular RNAs including those that encode for proteins are produced by the enzyme RNA Polymerase II. In this work, we have defined a new role for the phosphatase Rtr1 in the regulation of RNA Polymerase II progression from the start of transcription to the 3’ end of the gene where the nascent RNA from protein-coding genes is typically cleaved and polyadenylated. Deletion of the gene that encodes RTR1 leads to changes in the interactions between RNA polymerase II and the termination machinery. Rtr1 loss also causes early termination of RNA Polymerase II at many of its target gene types, including protein coding genes and noncoding RNAs. Evidence suggests that the premature termination observed in RTR1 knockout cells occurs through the termination factor and RNA binding protein Nrd1 and its binding partner Nab3. Deletion of RRP6, a known component of the Nrd1-Nab3 termination coupled RNA degradation pathway, is epistatic to RTR1 suggesting that Rrp6 is required to terminate and/or degrade many of the noncoding RNAs that have increased turnover in RTR1 deletion cells. These findings suggest that Rtr1 normally promotes elongation of RNA Polymerase II transcripts through prevention of Nrd1-directed termination.

Summary The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription.

NusA is an essential protein that binds to RNA polymerase (RNAP) and also to the nascent RNA, and influences transcription by inducing pausing and facilitating the process of transcription termination / antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that as both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA-binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlapping nut (NusA-binding site) and rut (Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at the rut site that inhibits the RNA release process of the latter. High density tiling micro-array profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions are up-regulated. Interestingly, majority of these genes were also up-regulated when the Rho function was compromised. These are strong evidences for the existence of NusA-binding sites in different operons which are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination.

International audience; Factor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Rho is nearly ubiquitous in bacteria, but the extent to which its physiological functions are conserved throughout the different phyla remains unknown. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. However, the rather comprehensive knowledge about the general mechanisms of action and activities of Rho based on the E. coli paradigm cannot be directly extrapolated to other bacteria. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similarities and differences of the activities of Rho in various bacterial species.

NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the transcription of ribosomal and actively-translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non- canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed- ring, catalytically-active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation, and further explains how Rho is excluded from translationally-competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor, and establishes how the innate sequence-bias of a termination factor can be modulated to silence pervasive, aberrant transcription.

Widely transcribed compact genomes must cope with the major challenge of frequent overlapping or concurrent transcription events. Efficient and timely transcription termination is crucial to control pervasive transcription and prevent transcriptional interference. In yeast, transcription termination of RNA polymerase II (RNAPII) occurs via two possible pathways that both require recognition of termination signals on nascent RNA by specific factors. We describe here an additional mechanism of transcription termination for RNAPII and demonstrate its biological significance. We show that the transcriptional activator Reb1p bound to DNA is a roadblock for RNAPII, which pauses and is ubiquitinated, thus triggering termination. Reb1p-dependent termination generates a class of cryptic transcripts that are degraded in the nucleus by the exosome. We also observed transcriptional interference between neighboring genes in the absence of Reb1p. This work demonstrates the importance of roadblock termination for controlling pervasive transcription and preventing transcription through gene regulatory regions.

In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA−DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991–1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.

In budding yeast, the 3′ end processing of mRNA and the coupled termination of transcription by RNAPII requires the CF IA complex. We have earlier demonstrated a role for the Clp1 subunit of this complex in termination and promoter-associated transcription of CHA1. To assess the generality of the observed function of Clp1 in transcription, we tested the effect of Clp1 on transcription on a genomewide scale using the Global Run-On-Seq (GRO-Seq) approach. GRO-Seq analysis showed the polymerase reading through the termination signal in the downstream region of highly transcribed genes in a temperature-sensitive mutant of Clp1 at elevated temperature. No such terminator readthrough was observed in the mutant at the permissive temperature. The poly(A)-independent termination of transcription of snoRNAs, however, remained unaffected in the absence of Clp1 activity. These results strongly suggest a role for Clp1 in poly(A)-coupled termination of transcription. Furthermore, the density of antisense transcribing polymerase upstream of the promoter region exhibited an increase in the absence of Clp1 activity, thus implicating Clp1 in promoter directionality. The overall conclusion of these results is that Clp1 plays a general role in poly(A)-coupled termination of RNAPII transcription and in enhancing promoter directionality in budding yeast.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific "readers." Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4- or phospho-Ser2-bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2-modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks.

RfaH activates horizontally acquired operons that encode lipopolysaccharide core components, pili, toxins, and capsules. Unlike its paralog NusG, which potentiates Rho-mediated silencing, RfaH strongly inhibits Rho. RfaH is recruited to its target operons via a network of contacts with an elongating RNA polymerase (RNAP) and a specific DNA element called ops to modify RNAP into a pause- and NusG-resistant state. rfaH null mutations confer hypersensitivity to antibiotics and detergents, altered susceptibility to bacteriophages, and defects in virulence. Here, we carried out a selection for suppressors that restore the ability of a ΔrfaH mutant Escherichia coli strain to grow in the presence of sodium dodecyl sulfate. We isolated rho, rpoC, and hns suppressor mutants with changes in regions previously shown to be important for their function. In addition, we identified mutants with changes in an unstructured region that connects the primary RNA-binding and helicase domains of Rho. The connector mutants display strong defects in vivo, consistent with their ability to compensate for the loss of RfaH, and act synergistically with bicyclomycin (BCM), which has been recently shown to inhibit Rho transformation into a translocation-competent state. We hypothesize that the flexible connector permits the reorientation of Rho domains and serves as a target for factors that control the motor function of Rho allosterically. Our results, together with the existing data, support a model in which the connector segment plays a hitherto overlooked role in the regulation of Rho-dependent termination., IMPORTANCE The transcription termination factor Rho silences foreign DNA, reduces antisense transcription, mediates surveillance of mRNA quality, and maintains genome integrity by resolving transcription-replication collisions and deleterious R loops. Upon binding to RNA, Rho undergoes a rate-limiting transition from an open “lock washer” state to a closed ring capable of processive translocation on, and eventually the release of, the nascent transcript. Recent studies revealed that Rho ligands, including its cofactor NusG and inhibitor bicyclomycin, control the ring dynamics allosterically. In this work, we used a genetic selection for suppressors of RfaH, a potent inhibitor of Rho, to isolate a new class of mutations in a flexible region that connects the primary RNA-binding and ATPase/translocase domains of Rho. We propose that the connector is essential for the modulation of Rho activity by different RNA sequences and accessory proteins.

λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a λN–NusA–NusB–NusE–nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and λN, validated by crosslinking/mass spectrometry. Due to intrinsic disorder, λN can act as a multiprotein/RNA interaction hub, which, together with nut site RNA, arranges NusA, NusB and NusE into a triangular complex. This complex docks via the NusA N-terminal domain and the λN C-terminus next to the RNA exit channel on RNA polymerase. Based on the structures, comparative crosslinking analyses and structure-guided mutagenesis, we hypothesize that λN mounts a multipronged strategy to reprogram the transcriptional machinery, which may include (1) the λN C terminus clamping the RNA exit channel, thus stabilizing the DNA:RNA hybrid; (2) repositioning of NusA and RNAP elements, thus redirecting nascent RNA and sequestering the upstream branch of a terminator hairpin; and (3) hindering RNA engagement of termination factor ρ and/or obstructing ρ translocation on the transcript. Structural characterization sheds mechanistic insights behind λN-dependent processive antitermination.

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3′ end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3′ end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Many RNA-binding proteins possess domains with a biased amino acid content. A common property of these low complexity domains (LCDs) is that they assemble into an ordered amyloid form, juxtaposing RNA recognition motifs in a subcellular compartment in which RNA metabolism is focused. Yeast Nab3 is one such protein that contains RNA-binding domains and a low complexity, glutamine/proline-rich, prion-like domain that can self-assemble. Nab3 also contains a region of structural homology to human hnRNP-C that resembles a leucine zipper which can oligomerize. Here we show that the LCD and the human hnRNP-C homology domains of Nab3 were experimentally separable, as cells were viable with either segment, but not when both were missing. In exploiting the lethality of deleting these regions of Nab3, we were able to test if heterologous prion-like domains known to assemble into amyloid, could substitute for the native sequence. Those from the hnRNP-like protein Hrp1, the canonical prion Sup35, or the epsin-related protein Ent2, could rescue viability and enable the new Nab3 chimeric protein to support transcription termination. Other low complexity domains from RNA-binding, termination-related proteins or a yeast prion, could not. As well, an unbiased genetic selection revealed a new protein sequence that could rescue the loss of Nab3’s essential domain via multimerization. This new sequence and Sup35’s prion domain could also rescue the lethal loss of Hrp1’s prion-like domain when substituted for it. This suggests there are different cross-functional classes of amyloid-forming LCDs and that appending merely any assembly-competent LCD to Nab3 does not restore function or rescue viability. The analysis has revealed the functional complexity of LCDs and provides a means by which the differing classes of LCD can be dissected and understood.

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks., Author summary Bacillus subtilis is a widely used model to study cell differentiation in the bacterial world. This soil-dwelling bacterium can engage in several alternative developmental programs, which generate distinct cell types adapted to different lifestyles, to cope with its complex and changing natural environment. The underlying differentiation control mechanisms are interconnected and tightly regulated, because these physiological and morphological cellular states are mutually exclusive and a correct choice is crucial. Here, we describe a previously unrecognized mechanism that regulates cell fate decisions in B. subtilis. It is based on the elements of pervasive genome-wide transcription controlled by the termination factor Rho. Pervasive transcription originating from non-defined or cryptic signals is spread throughout bacterial transcriptomes, but its physiological role is not yet well understood. We show that the elements of the Rho-controlled transcriptome affect specific developmental programs in B. subtilis: cell motility, biofilm formation, and sporulation, by directly or indirectly targeting expression of the key factors of cellular differentiation. Our results illuminate how Rho plays a prominent role in complex and organism-specific regulatory networks by controlling pervasive transcription. These findings rank Rho among the global transcriptional regulators of B. subtilis and invite systematic exploration of its role in other microorganisms.

Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.

Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5′ end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination.

Ess1 is a prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II. Ess1 works by catalyzing the cis/trans conversion of pSer5–Pro6 bonds, and to a lesser extent pSer2–Pro3 bonds, within the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA pol II. Ess1 is conserved in organisms ranging from yeast to humans. In budding yeast, Ess1 is essential for growth and is required for efficient transcription initiation and termination, RNA processing, and suppression of cryptic transcription. In mammals, Ess1 (called Pin1) functions in a variety of pathways, including transcription, but it is not essential. Recent work has shown that Ess1 coordinates the binding and release of CTD-binding proteins that function as co-factors in the RNA pol II complex. In this way, Ess1 plays an integral role in writing (and reading) the so-called CTD code to promote production of mature RNA pol II transcripts including non-coding RNAs and mRNAs.

Anti-Q is a small RNA encoded on pCF10, an antibiotic resistance plasmid of Enterococcus faecalis, which negatively regulates conjugation of the plasmid. In this study we sought to understand how Anti-Q is generated relative to larger transcripts of the same operon. We found that Anti-Q folds into a branched structure that functions as a factor-independent terminator. In vitro and in vivo, termination is dependent on the integrity of this structure as well as the presence of a 3' polyuridine tract, but is not dependent on other downstream sequences. In vitro, terminated transcripts are released from RNA polymerase after synthesis. In vivo, a mutant with reduced termination efficiency demonstrated loss of tight control of conjugation function. A search of bacterial genomes revealed the presence of sequences that encode Anti-Q-like RNA structures. In vitro and in vivo experiments demonstrated that one of these functions as a terminator. This work reveals a previously unappreciated flexibility in the structure of factor-independent terminators and identifies a mechanism for generation of functional small RNAs; it should also inform annotation of bacterial sequence features, such as terminators, functional sRNAs, and operons.

Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.

Nab3 is an RNA-binding protein whose function is important for terminating transcription by RNA polymerase II. It co-assembles with Nrd1, and the resulting heterodimer of these heterogeneous nuclear ribonucleoprotein-C (hnRNP)-like proteins interacts with the nascent transcript and RNA polymerase II. Previous genetic analysis showed that a short carboxyl-terminal region of Nab3 is functionally important for termination and is located far from the Nab3 RNA recognition domain in the primary sequence. The domain is structurally homologous to hnRNP-C from higher organisms. Here we provide biochemical evidence that this short region is sufficient to enable self-assembly of Nab3 into a tetrameric form in a manner similar to the cognate region of human hnRNP-C. Within this region, there is a stretch of low complexity protein sequence (16 glutamines) adjacent to a putative α-helix that potentiates the ability of the conserved region to self-assemble. The glutamine stretch and the final 18 amino acids of Nab3 are both important for termination in living yeast cells. The findings herein describe an additional avenue by which these hnRNP-like proteins can polymerize on target transcripts. This process is independent of, but acts in concert with, the interactions of the proteins with RNA and RNA polymerase and extends the relationship of Nab3 as a functional orthologue of a higher eukaryotic hnRNP.

Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (∼20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1–Nrd1–Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.

The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease., Author Summary RNA polymerase (RNAP) III is a multisubunit enzyme that synthesizes large amounts of transfer RNAs and a few other abundant short structural RNAs essential for the proliferation of all eukaryotic cells. To meet this demand, RNAP III must repeatedly initiate and terminate RNA synthesis with high efficiency, up to one million times on each tRNA gene per cell doubling. Much more research has been done on initiation than on termination. We performed a genetic screen focused on Rpc1, the largest RNAP III subunit, after subjecting it to random mutagenesis and identifying single amino acid substitutions that specifically affected its ability to terminate. Over 120 mutations were isolated and mapped onto a recently published, near-atomic resolution structure of RNAP III in synthesis mode. Mutations were concentrated in mobile elements of the active center of the enzyme, the bridge helix and the trigger loop, as well as regions that help recruit the RNA 3' cleavage domain of the C11 subunit which transiently interacts with the active center. The data provide evidence that RNAP III controls termination via its active center. Biochemical characterization demonstrated that a mutant RNAP III synthesizes RNA at a higher rate than wild type and also synthesizes more RNA in vivo.

The poly(A) (pA) signal possesses a dual function in 3′ end processing of pre-mRNA and in transcriptional termination of RNA polymerase II (Pol II) for most eukaryotic protein-coding genes. A key protein factor in yeast and Drosophila Pol II transcriptional termination is the 3′-end processing factor, Pcf11. In vitro studies suggest that Pcf11 is capable of promoting the dissociation of Pol II elongation complexes from DNA. Moreover, several mutant alleles of yeast Pcf11 effect termination in vivo. However, functions of human Pcf11 (hPcf11) in Pol II termination have not been explored. Here we show that depletion of hPcf11 from HeLa cells reduces termination efficiency. Furthermore, we provide evidence that hPcf11 is required for the efficient degradation of the 3′ product of pA site cleavage. Finally, we show that these functions of hPcf11 require an intact pA signal.

Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed ofRhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of thecleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

Enhancement of transcription by a splicing-competent intron is an evolutionarily conserved feature among eukaryotes. The molecular mechanism underlying the phenomenon, however, is not entirely clear. Here we show that the intron is an important regulator of promoter directionality. Employing strand-specific transcription run-on (TRO) analysis, we show that the transcription of mRNA is favored over the upstream anti-sense transcripts (uaRNA) initiating from the promoter in the presence of an intron. Mutation of either the 5′ or 3′ splice site resulted in the reversal of promoter directionality, thereby suggesting that it is not merely the 5′ splice site but the entire splicing-competent intron that regulates transcription directionality. ChIP analysis revealed the recruitment of termination factors near the promoter region in the presence of an intron. Removal of intron or the mutation of splice sites adversely affected the promoter localization of termination factors. We have earlier demonstrated that the intron-mediated enhancement of transcription is dependent on gene looping. Here we show that gene looping is crucial for the recruitment of termination factors in the promoter-proximal region of an intron-containing gene. In a looping-defective mutant, despite normal splicing, the promoter occupancy of factors required for poly(A)-dependent termination of transcription was compromised. This was accompanied by a concomitant loss of transcription directionality. On the basis of these results, we propose that the intron-dependent gene looping places the terminator-bound factors in the vicinity of the promoter region for termination of the promoter-initiated upstream antisense transcription, thereby conferring promoter directionality., Author Summary Eukaryotic genes differ from their prokaryotic counterparts in having intervening non-coding sequences called introns. The precise biological role of introns in eukaryotic systems remains unclear even more than forty years after their initial discovery. One function of intron that has been remarkably conserved during evolution is their ability to enhance the transcription of genes that harbor them. How does the intron regulate transcription, however, is not known. Here we show that the intron enhances gene expression by affecting direction of the promoter-initiated transcription. In the presence of an intron, polymerase tends to transcribe the downstream coding region producing mRNA, while in the absence of a splicing-competent intron polymerase starts transcribing promoter upstream region producing upstream antisense RNA (uaRNA). Intron-mediated promoter directionality was dependent on gene looping, which is the interaction off the promoter and terminator region of a gene in a transcription-dependent manner. We show that the intron-dependent gene looping facilitates the recruitment of termination factors in the promoter-proximal region. The recruited termination factors stop uaRNA synthesis thereby conferring directionality to the promoter-bound polymerase.

Base J, β-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In Trypanosoma brucei, J is enriched, along with histone H3 variant (H3.V), at sites involved in RNA Polymerase (RNAP) II termination and telomeric sites involved in regulating variant surface glycoprotein gene (VSG) transcription by RNAP I. Reduction of J in T. brucei indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the H3.V knockout. mRNA-seq indicates gene derepression includes VSG genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in T. brucei, depending on chromatin context (and transcribing polymerase). As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination., Author Summary Trypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. Because of this genome arrangement, it is thought that all gene regulation in T. brucei occurs after transcription at the level of RNA (processing, stability, and translation). We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J leads to read-through transcription and the expression of downstream genes. We now find that H3.V also promotes termination prior to the end of gene clusters, thus regulating the transcription of specific genes. Additionally, H3.V inhibits transcription of siRNA producing loci. Our data suggest H3.V and base J are utilized for regulating gene expression via terminating transcription within polycistronic gene arrays and regulating the synthesis of siRNAs in trypanosomes. These findings significantly expand our understanding of epigenetic regulatory mechanisms underlying transcription termination in eukaryotes, including divergent organisms that utilize polycistronic transcription, providing the first example of a histone variant that promotes transcription termination.

6S RNA is a bacterial transcriptional regulator, which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ70-containing RNA polymerase. This inhibitory RNA polymerase~6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs. Here, we used limited hydrolysis with structure-specific RNases and in-line probing of isolated 6S RNA and 6S RNA~pRNA complexes to investigate the molecular details leading to the release reaction. Our results indicate that pRNA transcription induces the refolding of the 6S RNA secondary structure by disrupting part of the closing stem (conserved sequence regions CRI and CRIV) and formation of a new hairpin (conserved sequence regions CRIII and CRIV). Comparison of the dimethylsulfate modification pattern of 6S RNA in living cells at stationary growth and during outgrowth confirmed the conformational change observed in vitro. Based on our results, a model describing the individual steps of the release reaction is presented.

How eukaryotic RNA polymerases switch from elongation to termination is unknown. Pol III subunits Rpc53 and Rpc37 (C53/37) form a heterodimer homologous to TFIIFβ/α. C53/37 promotes efficient termination and together with C11 also mediates pol III recycling in vitro. We previously developed Schizosaccharomyces pombe strains that report on two pol III termination activities: RNA oligo(U) 3'-end cleavage, and terminator readthrough. We randomly mutagenized C53 and C37 and isolated many C37 mutants with terminator readthrough but no comparable C53 mutants. The majority of C37 mutants have strong phenotypes with up to 40% readthrough and map to a C-terminal tract previously localized near Rpc2p in the pol III active center while a minority represent a distinct class with weaker phenotype, less readthrough and 3'-oligo(U) lengthening. Nascent pre-tRNAs released from a terminator by C37 mutants have shorter 3'-oligo(U) tracts than in cleavage-deficient C11 double mutants indicating RNA 3'-end cleavage during termination. We asked whether termination deficiency affects transcription output in the mutants in vivo both by monitoring intron-containing nascent transcript levels and (14)C-uridine incorporation. Surprisingly, multiple termination mutants have no decrease in transcript output relative to controls. These data are discussed in context of current models of pol III transcription.

The conventional model of Rho-dependent transcription termination in bacteria requires RNA-dependent translocase activity of the termination factor Rho as well as many kinetically controlled steps to execute efficient RNA release from the transcription elongation complex (EC). The involvement of the kinetically controlled steps, such as RNA binding, translocation and RNA release from the EC, means that this termination process must be kinetically coupled to the transcription elongation process. The existence of these steps in vivo has not previously been delineated in detail. Moreover, the requirement for translocase activity in Rho-dependent termination has recently been questioned by a radical view, wherein Rho binds to the elongating RNA polymerase (RNAP) prior to loading onto the mRNA. Using growth assays, microarray analyses and reporter-based transcription termination assays in vivo, we showed that slowing of the transcription elongation rate by using RNAP mutants (rpoB8 and rpoB3445) and growth of the strains in minimal medium suppressed the termination defects of five Rho mutants, three NusG mutants defective for Rho binding and the defects caused by two Rho inhibitors, Psu and bicyclomycin. These results established the existence of kinetically controlled steps in the in vivo Rho-dependent termination process and further reinforced the importance of 'kinetic coupling' between the two molecular motors, Rho and RNAP, and also argue strongly that the Rho translocation model is an accurate representation of the in vivo situation. Finally, these results indicated that one of the major roles of NusG in in vivo Rho-dependent termination is to enhance the speed of RNA release from the EC.

RNA polymerase is a ratchet machine that oscillates between productive and backtracked states at numerous DNA positions. The amount of backtracking (reversible sliding of the enzyme along DNA and RNA) varies from one to many nucleotides. Since its first description 15 years ago, backtracking has been implicated in a plethora of critical processes in bacteria and eukaryotic cells. Here we review the most fundamental roles of this phenomenon in controlling transcription elongation, pausing, termination, fidelity, and genome instability. We also discuss recent progress in understanding the structural and mechanistic properties of the backtracking process.

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 'tail' forms an α-helical multimerization domain that helps assemble it onto an RNA substrate.

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser 5 -phosphorylated (Ser 5 -P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W , a mutation that impairs an interaction between Sen1 and the Ser 2 -phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser 2 -P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser 5 - to the Ser 2 -phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

The pervasiveness of RNA synthesis in eukaryotes is largely the result of RNA polymerase II (Pol II)-mediated transcription, and termination of its activity is necessary to partition the genome and maintain the proper expression of neighbouring genes. Despite its ever-increasing biological significance, transcription termination remains one of the least understood processes in gene expression. However, recent mechanistic studies have revealed a striking convergence among several overlapping models of termination, including the poly(A)- and Sen1-dependent pathways, as well as new insights into the specificity of Pol II termination among its diverse gene targets. Broader knowledge of the role of Pol II carboxy-terminal domain phosphorylation in promoting alternative mechanisms of termination has also been gained.

Termination signals induce rapid and irreversible dissociation of the nascent transcript from RNA polymerase. Terminators at the end of genes prevent unintended transcription into the downstream genes, whereas terminators in the upstream regulatory leader regions adjust expression of the structural genes in response to metabolic and environmental signals. Premature termination within an operon leads to potentially deleterious defects in the expression of the downstream genes, but also provides an important surveillance mechanism. This Review discusses the actions of bacterial and phage antiterminators that allow RNA polymerase to override a terminator when the circumstances demand it.

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.

Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.

Rapid diagnostic identification of the human H5 influenza virus is a strategic cornerstone for outbreak prevention. We recently reported a method for direct detection of viral RNA from a highly pathogenic human H5 influenza strain (A/Hanoi/30408/2005(H5N1)), which necessarily was transcribed in vitro from non-viral sources. This article provides an in-depth analysis of the reaction conditions for in vitro transcription (IVT) of full-length influenza H5 RNA, which is needed for diagnostic RNA production, for the T7 and SP6 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5' SP6 promoter and 3' restriction site (BsmBI) showed that three sequence-verified bands at 1,776, 784, and 591 bases were consistently produced, whereas only one 1,776-base band was expected. These fragments were not observed in H1 or H3 influenza RNA transcribed under similar conditions. A reverse complement of the sequence produced only a single band at 1,776 bases, which suggested either self-cleavage or early termination. Aliquots of the IVT reaction were quenched with EDTA to track the generation of the bands over time, which maintained a constant concentration ratio. The H5 sequence was cloned with T7 and SP6 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the vRNA direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the SP6 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length vRNA transcripts than the SP6 system did under similar reaction conditions. By sequencing we identified a type II RNA hairpin loop terminator, which forms in a transcription direction-dependent fashion. Variation of the magnesium concentration produced the greatest impact on termination profiles, where some reaction mixtures were unable to produce full-length transcripts. Optimized conditions are presented for the T7 and SP6 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza vRNA.

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism controlled by the trp RNA-binding attenuation protein (TRAP). TRAP binds to 11 (G/U)AG repeats in the trp leader transcript and prevents formation of an antiterminator, which allows formation of an intrinsic terminator (attenuator). Previously, formation of the attenuator RNA structure was believed to be solely responsible for signaling RNA polymerase (RNAP) to halt transcription. However, base substitutions that prevent formation of the antiterminator, and thus allow the attenuator structure to form constitutively, do not result in efficient transcription termination. The observation that the attenuator requires the presence of TRAP bound to the nascent RNA to cause efficient transcription termination suggests TRAP has an additional role in causing termination at the attenuator. We show that the trp attenuator is a weak intrinsic terminator due to low GC content of the hairpin stem and interruptions in the U-stretch following the hairpin. We also provide evidence that termination at the trp attenuator requires forward translocation of RNA polymerase and that TRAP binding to the nascent transcript can induce this activity.

The pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. A systematic study previously performed revealed the presence of nine major transcripts, the expression of which was differentially and temporally regulated over the growth cycle of S. islandicus. In this study, two new transcripts were identified. Then, 3' termini of all the RNAs were mapped using adaptor RT-PCR and RNase protection assays, and termination/arrest positions were identified for each transcript. The majority of the identified ending positions were located in the close vicinity of a T-rich sequence and this was consistent with termination signals identifiable for most of archaeal genes. Furthermore, termination also occurred at locations where a T-track sequence was absent but a stem-loop structure could be formed. We propose that an alternative mechanism based on secondary RNA structures and counter-transcripts might be responsible for the transcription termination at these T-track-minus loci in the closely spaced pSSVx genes.

Maturation of some snoRNAs is dependent on RNase III-like endonuclease-mediated transcript cleavage, which serves as an entry for the nuclear exosome complex that trims the transcript at the 3′-end. Sequence deletions suggest this cleavage in the U3 snoRNA transcripts of Schizosaccharomyces pombe can induce transcript termination. Using mutational analyses, we demonstrate that the degree of cleavage correlates closely with both RNA maturation and transcript termination. We also show that the RNase III-like endonuclease, Pac1, and the nuclear 5′-exonuclease, Dhp1p, are essential for RNA production and transcript termination, supporting a “reversed torpedoes” model in which the endonuclease cut allows 5′- and 3′-exonuclease activities access to the transcript, leading simultaneously to transcript termination in one direction and RNA maturation in the other.

© 2010 Future Medicine Ltd, Transcription of protein-coding genes by RNA polymerase II is a repetitive, cyclic process that enables synthesis of multiple RNA molecules from the same template. The transcription cycle consists of three main stages, initiation, elongation and termination. Each of these phases is intimately coupled to a specific step in pre-mRNA processing; 5´ capping, splicing and 3´-end formation, respectively. In this article, we discuss the recent concept that cotranscriptional checkpoints operate during mRNA biogenesis to ensure that nonfunctional mRNAs with potentially deleterious effects for the cell are not produced or exported to the cytoplasm for translation., The authors’ laboratory is supported by grants from Fundação para a Ciência e Tecnologia, Portugal (PTDC/BIA-BCM/101575/2008) and the European Commission (LSHG-CT-2005–518238, EURASNET). Sérgio F. de Almeida is supported by a fellowship from Fundação para a Ciência e Tecnologia (SFRH/BPD/34679/2007).

Transcription and Translation in Train In bacteria, translation of messenger RNA into proteins by the ribosome usually begins soon after the ribosome binding site emerges from RNA polymerase. Now there is evidence for direct coupling between transcription and translation in bacteria. Proshkin et al. (p. 504 ; see the Perspective by Roberts ) show that the trailing ribosome controls the rate of transcription by preventing RNA polymerase from spontaneous backtracking, which allows precise adjustment of transcriptional yield to translational needs under various growth conditions. Burmann et al. (p. 501 ; see the Perspective by Roberts ) provide a potential mechanism for coupling by showing that the transcription factor NusG, which binds RNA polymerase through its amino-terminal domain, competitively binds either a ribosomal protein or the Rho transcription termination factor through its carboxy-terminal domain. Rho binding might occur after release of the ribosome from messenger RNA, thus linking termination of transcription and translation.

The maturation of many small nuclear RNAs is dependent on RNase III-like endonuclease mediated cleavage, which generates a loading site for the exosome complex that trims the precursor at its 3' end. Using a temperature sensitive Pac1 nuclease, here we show that the endonuclease cleavage is equally important in terminating the transcription of the U2 snRNA in Schizosaccharomyces pombe. Using a temperature sensitive Dhp1p 5'-->3' exonuclease, we demonstrate that it also is an essential component of the termination pathway. Taken together the results support a "reversed torpedoes" model for the termination and maturation of the U2 snRNA; the Pac1 endonuclease cleavage provides entry sites for the 3' and 5' exonuclease activities, leading to RNA maturation in one direction and transcript termination in the other.

Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria. Although Rho was the first termination factor to be discovered, the actual mechanism by which it reaches and disrupts the elongation complex (EC) is unknown. Here we show that the termination-committed Rho molecule associates with RNA polymerase (RNAP) throughout the transcription cycle; that is, it does not require the nascent transcript for initial binding. Moreover, the formation of the RNAP-Rho complex is crucial for termination. We show further that Rho-dependent termination is a two-step process that involves rapid EC inactivation (trap) and a relatively slow dissociation. Inactivation is the critical rate-limiting step that establishes the position of the termination site. The trap mechanism depends on the allosterically induced rearrangement of the RNAP catalytic centre by means of the evolutionarily conserved mobile trigger-loop domain, which is also required for EC dissociation. The key structural and functional similarities, which we found between Rho-dependent and intrinsic (Rho-independent) termination pathways, argue that the allosteric mechanism of termination is general and likely to be preserved for all cellular RNAPs throughout evolution.