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In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3'-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation., Competing Interests: The authors have declared that no competing interests exist.

Many RNA polymerases terminate transcription using allosteric/intrinsic mechanisms, whereby protein alterations or nucleotide sequences promote their release from DNA. RNA polymerase II (Pol II) is somewhat different based on its behavior at protein-coding genes where termination additionally requires endoribonucleolytic cleavage and subsequent 5'→3' exoribonuclease activity. The Pol-II-transcribed small nuclear RNAs (snRNAs) also undergo endoribonucleolytic cleavage by the Integrator complex, which promotes their transcriptional termination. Here, we confirm the involvement of Integrator but show that Integrator-independent processes can terminate snRNA transcription both in its absence and naturally. This is often associated with exosome degradation of snRNA precursors that long-read sequencing analysis reveals as frequently terminating at T-runs located downstream of some snRNAs. This finding suggests a unifying vulnerability of RNA polymerases to such sequences given their well-known roles in terminating Pol III and bacterial RNA polymerase., Competing Interests: Declaration of Interests The authors declare no competing interests., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of erh1 up-regulates the phosphate homeostasis gene pho1 , which is normally repressed by transcription in cis of a 5' flanking prt lncRNA. Here we present evidence that de-repression of pho1 by e rh1 Δ is achieved through precocious 3'-processing/termination of prt lncRNA synthesis, to wit: (i) erh1 Δ does not affect the activity of the prt or pho1 promoters per se; (ii) de-repression by erh1 Δ depends on CPF (cleavage and polyadenylation factor) subunits Ctf1, Dis2, Ssu72, Swd22, and Ppn1 and on termination factor Rhn1; (iii) de-repression requires synthesis by the Asp1 IPP kinase of inositol 1-pyrophosphates (1-IPPs); (iv) de-repression is effaced by mutating Thr4 of the RNA polymerase II CTD to alanine; and (v) erh1 Δ exerts an additive effect on pho1 de-repression in combination with mutating CTD Ser7 to alanine and with deletion of the IPP pyrophosphatase Aps1. These findings point to Erh1 as an antagonist of lncRNA termination in the prt-pho1 axis. In contrast, in mmi1 Δ cells there is a reduction in pho1 mRNA and increase in the formation of a prt-pho1 read-through transcript, consistent with Mmi1 being an agonist of prt termination. We envision that Erh1 acts as a brake on Mmi1's ability to promote CPF-dependent termination during prt lncRNA synthesis. Consistent with this idea, erh1 Δ de-repression of pho1 was eliminated by mutating the Mmi1-binding sites in the prt lncRNA., (© 2020 Schwer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing., (© 2020 Eaton et al.; Published by Cold Spring Harbor Laboratory Press.)

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.

Stop-codon read-through refers to the phenomenon that a ribosome goes past the stop codon and continues translating into the otherwise untranslated region (UTR) of a transcript. Recent ribosome-profiling experiments in eukaryotes uncovered widespread stop-codon read-through that also varies among tissues, prompting the adaptive hypothesis that stop-codon read-through is an important, regulated mechanism for generating proteome diversity. Here we propose and test a competing hypothesis that stop-codon read-through arises mostly from molecular errors and is largely nonadaptive. The error hypothesis makes distinct predictions about the probability of read-through, frequency of sequence motifs for read-through, and conservation of the read-through region, each of which is supported by genome-scale data from yeasts and fruit flies. Thus, except for the few cases with demonstrated functions, stop-codon read-through is generally nonadaptive. This finding, along with other molecular errors recently quantified, reveals a much less precise or orderly cellular life than is commonly thought., Competing Interests: The authors have declared that no competing interests exist.

Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate transcription without transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II transcription bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion.

In bacteria, transcription-coupled repair of DNA lesions initiates after the Mfd protein removes RNA polymerases (RNAPs) stalled at the lesions. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. Here, we show that Rho dislodges the stalled RNAPs at DNA lesions. Strains defective in both Rho and Mfd are susceptible to DNA-damaging agents and are inefficient in repairing or propagating UV-damaged DNA. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We conclude that Rho-dependent termination recycles stalled RNAPs, which might facilitate DNA repair and other DNA-dependent processes essential for bacterial cell survival. We surmise that Rho might compete with, or augment, the Mfd function.

Termination of RNA polymerase II (RNAPII) transcription is a fundamental step of gene expression that is critical for determining the borders between genes. In budding yeast, termination at protein-coding genes is initiated by the cleavage/polyadenylation machinery, whereas termination of most noncoding RNA (ncRNA) genes occurs via the Nrd1-Nab3-Sen1 (NNS) pathway. Here, we find that NNS-like transcription termination is not conserved in fission yeast. Rather, genome-wide analyses show global recruitment of mRNA 3' end processing factors at the end of ncRNA genes, including snoRNAs and snRNAs, and that this recruitment coincides with high levels of Ser2 and Tyr1 phosphorylation on the RNAPII C-terminal domain. We also find that termination of mRNA and ncRNA transcription requires the conserved Ysh1/CPSF-73 and Dhp1/XRN2 nucleases, supporting widespread cleavage-dependent transcription termination in fission yeast. Our findings thus reveal that a common mode of transcription termination can produce functionally and structurally distinct types of polyadenylated and non-polyadenylated RNAs.

Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 dephosphorylation by the conserved PP1 phosphatase Dis2, which also dephosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and dephosphorylation of Pol II C-terminal domain., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 synthesis 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3' direction several hundred kilobases downstream of genes. The YFFF mutant shows reduced Pol II at promoter-proximal pause sites, a loss of interaction with the Mediator and Integrator complexes, and impaired recruitment of these complexes to chromatin. Consistent with these observations, Pol II loading at enhancers and maturation of snRNAs are altered in the YFFF context genome-wide. We conclude that tyrosine1 residues of the CTD control termination of transcription by Pol II., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Transcription termination determines the ends of transcriptional units and thereby ensures the integrity of the transcriptome and faithful gene regulation. Studies in yeast and human cells have identified the exoribonuclease XRN2 as a key termination factor for protein-coding genes. Here we performed a genome-wide investigation of RNA polymerase II (Pol II) transcription termination in XRN2-deficient Caenorhabditis elegans and observed two distinct modes of termination. Although a subset of genes requires XRN2, termination of other genes appears both independent of, and refractory to, XRN2. XRN2 independence is not merely a consequence of failure to recruit XRN2, since XRN2 is present on-and promotes Pol II accumulation near the polyadenylation sites of-both gene classes. Unexpectedly, promoters instruct the choice of termination mode, but XRN2-independent termination additionally requires a compatible region downstream from the 3' end cleavage site. Hence, different termination mechanisms may work with different configurations of Pol II complexes dictated by promoters., (© 2017 Miki et al.; Published by Cold Spring Harbor Laboratory Press.)

Termination of Saccharomyces cerevisiae RNA polymerase II (Pol II) transcripts occurs through two alternative pathways. Termination of mRNAs is coupled to cleavage and polyadenylation while noncoding transcripts are terminated through the Nrd1-Nab3-Sen1 (NNS) pathway in a process that is linked to RNA degradation by the nuclear exosome. Some mRNA transcripts are also attenuated through premature termination directed by the NNS complex. In this paper we present the results of nuclear depletion of the NNS component Nab3. As expected, many noncoding RNAs fail to terminate properly. In addition, we observe that nitrogen catabolite-repressed genes are upregulated by Nab3 depletion., (Copyright © 2017 American Society for Microbiology.)

The bacterial transcription elongation factor NusG stimulates the Rho-dependent transcription termination through a direct interaction with Rho. The mechanistic basis of NusG dependence of the Rho function is not known. Here, we describe Rho* mutants I168V, R221C/A, and P235H that do not require NusG for their termination function. These Rho* mutants have acquired new properties, which otherwise would have been imparted by NusG. A detailed analyses revealed that they have more stable interactions at the secondary RNA binding sites of Rho, which reduced the lag in initiating its ATPase as well as the translocase activities. These more stable interactions arose from the significant spatial re-orientations of the P, Q, and R structural loops of the Rho central channel. We propose that NusG imparts similar conformational changes in the central channel of Rho, yielding faster isomerization of the open to the closed hexameric states of the latter during its RNA-loading step. This acceleration stabilizes the Rho-RNA interactions at many terminators having suboptimal rut sites, thus making Rho-NusG interactions so essential in vivo Finally, identification of the NusG binding sites on the Rho hexamer led us to conclude that the former exerts its effect allosterically., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Human mitochondrial transcription termination occurs within the leu-tRNA gene and is mediated by the DNA binding protein MTERF1. The crystal structure of MTERF1 bound to the canonical termination sequence reveals a rare base flipping event that involves the eversion of three nucleotides. These nucleotides are stabilized by stacking interactions with three MTERF1 residues, which are essential not only for base flipping but also for termination activity. To further understand the mechanism of base flipping, we examined each of the individual stacking interactions in structural, energetic and functional detail. Individual substitutions of Arg162, Tyr288 and Phe243 have revealed unequal contributions to overall termination activity. Furthermore, our work identifies an important role for Phe322 in the base flipping mechanism and we demonstrate how Phe322 and Phe243 are important for coupling base flipping between the heavy and light strand DNA chains. We propose a stepwise model for the base flipping process that recapitulates our observations. Finally, we show that MTERF1 has the ability to accommodate alternate active conformations. The adaptability of base flipping has implications for MTERF1 function and for the putative function of MTERF1 at alternative binding sites in human mitochondria., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA, and restricted Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo., (© 2015 Grzechnik et al.; Published by Cold Spring Harbor Laboratory Press.)

The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site. Batch crystallization under oil was required to produce crystals of good quality for data collection. Crystals grew in space group P2₁, with unit-cell parameters a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°. The crystals diffracted to a resolution of 3.0 Å. The crystals were mosaic and required two or three cycles of annealing. This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination.

Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration targeting-sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3' end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disruption of histone mRNA processing, and polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites.

DNA with four guanine tracts can fold into G-quadruplexes that are targets of transcription regulation. We recently found that hybrid DNA:RNA G-quadruplexes (HQs) can form during in vitro transcription. However, it is unclear whether they can form in cells. Evidence is presented supporting their formation in plasmids in bacterial cells. The formation of the HQs is indicated by a unique pattern of prematurely terminated transcripts under two conditions where the RNA transcripts do or do not participate in G-quadruplex assembly and further supported by a number of chemical and biochemical analysis. HQs dominate over the intramolecular DNA G-quadruplexes (DQ) in mediating the transcription termination when both structures are able to form. These findings provide the first evidence of HQ formation in cells and suggest that the competition/conversion between HQ and DQ may regulate transcription and serve as drug target in pharmaceutical applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we investigated the possibility of increasing the level of transgene expression using DNA element that can terminate transcription.

Eukaryotic RNA polymerase III (pol III) transcribes short noncoding RNA genes such as those encoding tRNAs, 5S rRNA, U6 snRNA, and a few others. As compared to its pol II counterpart, Pol III has several advantages, including the relative simplicity, stability, and more direct connectivity of its transcription machinery. Only two transcription factor complexes, TFIIIB and TFIIIC, are required to faithfully initiate and direct multiple rounds of transcription by pol III. Moreover, in contrast to an intricate multipartite mechanism of pol II termination, pol III termination is extremely simple, responsive to a monopartite signal (oligo T stretch on the nontemplate DNA strand) and mediated by a stably associated termination subcomplex of three integral subunits (Arimbasseri et al. Transcription 4(6), 2013). This makes pol III a valuable model for dissecting intrinsic molecular mechanisms of eukaryotic transcription termination. In this chapter, we provide protocols we adapted to study the biochemistry of transcription termination by S. cerevisiae pol III.

Transcription elongation by multisubunit RNA polymerases (RNAPs) is processive, but neither uniform nor continuous. Regulatory events during elongation include pausing, backtracking, arrest, and transcription termination, and it is critical to determine whether the absence of continued synthesis is transient or permanent. Here we describe mechanisms to generate large quantities of stable archaeal elongation complexes on a solid support to permit (1) single-round transcription, (2) walking of RNAP to any defined template position, and (3) discrimination of transcripts that are associated with RNAP from those that are released to solution. This methodology is based on untagged proteins transcribing biotin- and digoxigenin-labeled DNA templates in association with paramagnetic particles.

The gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378: 318-327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3' ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5' ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galE-galT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5' ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3' ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5' portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Many horizontally acquired genes (xenogenes) in the bacterium Escherichia coli are maintained in a silent transcriptional state by the nucleoid-associated transcription regulatory protein H-NS. Recent evidence has shown that antibiotic-mediated inhibition of the transcription terminator protein Rho leads to de-repression of horizontally acquired genes, akin to a deletion of hns. The mechanism behind this similarity in outcomes between the perturbations of two distinct processes remains unclear. Using ChIP-seq of H-NS in wild-type cells, in addition to that in cells treated with bicyclomycin--a specific inhibitor of Rho, we show that bicyclomycin treatment leads to a decrease in binding signal for H-NS to the E. coli chromosome. Rho inhibition leads to RNA polymerase readthrough, which in principle could displace H-NS from the DNA, thus leading to transcriptional derepression of H-NS-silenced genes. Other possible mediators of the effect of Rho on H-NS are discussed. A possible positive feedback between Rho and H-NS might help reinforce xenogene silencing.

In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism. When intracellular tryptophan levels are high, the TRAP protein binds to the 5' leader region of the nascent trp mRNA and induces transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind and the operon is transcribed. Two competing RNA secondary structures termed the antiterminator and terminator (attenuator) can form in the leader region RNA. In prior attenuation models, the only role of TRAP binding was to alter the RNA secondary structure to allow formation of the attenuator, which has been thought function as an intrinsic transcription terminator. However, recent studies have shown that the attenuator is not an effective intrinsic terminator. From these studies it was not clear whether TRAP functions independently or requires the presence of the attenuator RNA structure. Hence we have further examined the role of the attenuator RNA in TRAP-mediated transcription termination. TRAP was found to cause efficient transcription termination in the trp leader region in vivo when the attenuator was mutated or deleted. However, TRAP failed to induce transcription termination at these mutant attenuators in a minimal in vitro transcription system with B. subtilis RNA polymerase. Further studies using this system showed that NusA as well as the timing of TRAP binding to RNA play a role in the observed differences in vivo and in vitro.

Nab3 and Nrd1 are yeast heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins that heterodimerize and bind RNA. Genetic and biochemical evidence reveals that they are integral to the termination of transcription of short non-coding RNAs by RNA polymerase II. Here we define a Nab3 mutation (nab3Δ134) that removes an essential part of the protein's C terminus but nevertheless can rescue, in trans, the phenotype resulting from a mutation in the RNA recognition motif of Nab3. This low complexity region of Nab3 appears intrinsically unstructured and can form a hydrogel in vitro. These data support a model in which multiple Nrd1-Nab3 heterodimers polymerize onto substrate RNA to effect termination, allowing complementation of one mutant Nab3 molecule by another lacking a different function. The self-association property of Nab3 adds to the previously documented interactions between these hnRNP-like proteins, RNA polymerase II, and the nascent transcript, leading to a network of nucleoprotein interactions that define a higher order Nrd1-Nab3 complex. This was underscored from the synthetic phenotypes of yeast strains with pairwise combinations of Nrd1 and Nab3 mutations known to affect their distinct biochemical activities. The mutations included a Nab3 self-association defect, a Nab3-Nrd1 heterodimerization defect, a Nrd1-polymerase II binding defect, and an Nab3-RNA recognition motif mutation. Although no single mutation was lethal, cells with any two mutations were not viable for four such pairings, and a fifth displayed a synthetic growth defect. These data strengthen the idea that a multiplicity of interactions is needed to assemble a higher order Nrd1-Nab3 complex that coats specific nascent RNAs in preparation for termination.

The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein that acts as a docking platform integrating signal transduction pathways to nucleic acid-related processes. Given that hnRNPK could be involved in other steps that compose gene expression the definition of its genome-wide occupancy is important to better understand its role in transcription and co-transcriptional processes. Here, we used chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to analyze the genome-wide hnRNPK-DNA interaction in colon cancer cell line HCT116. 9.1/3.6 and 7.0/3.4 million tags were sequenced/mapped, then 1809 and 642 hnRNPK binding sites were detected in quiescent and 30-min serum-stimulated cells, respectively. The inspection of sequencing tracks revealed inducible hnRNPK recruitment along a number of immediate early gene loci, including EGR1 and ZFP36, with the highest densities present at the transcription termination sites. Strikingly, hnRNPK knockdown with siRNA resulted in increased pre-RNA levels transcribed downstream of the EGR1 polyadenylation (A) site suggesting altered 3'-end pre-RNA degradation. Further ChIP survey of hnRNPK knockdown uncovered decreased recruitment of the 5'-3' exonuclease XRN2 along EGR1 and downstream of the poly(A) signal without altering RNA polymerase II density at these sites. Immunoprecipitation of hnRNPK and XRN2 from intact and RNase A-treated nuclear extracts followed by shotgun mass spectrometry revealed the presence of hnRNPK and XRN2 in the same complexes along with other spliceosome-related proteins. Our data suggest that hnRNPK may play a role in recruitment of XRN2 to gene loci thus regulating coupling 3'-end pre-mRNA processing to transcription termination.

Proper RNA polymerase II (Pol II) transcription termination is essential to generate stable transcripts, to prevent interference at downstream loci, and to recycle Pol II back to the promoter (1-3). As such, termination is an intricately controlled process that is tightly regulated by a variety of different cis- and trans-acting factors (4, 5). Although many eukaryotic termination factors have been identified to date, the details of the precise molecular mechanisms governing termination remain to be elucidated. We devised an in vitro transcription system to study specific Pol II termination. We show for the first time that the exonucleolytic Rat1·Rai1 complex can elicit the release of stalled Pol II in vitro and can do so in the absence of other factors. We also find that Rtt103, which interacts with the Pol II C-terminal domain (CTD) and with Rat1, can rescue termination activity of an exonucleolytically deficient Rat1 mutant. In light of our findings, we posit a model whereby functional nucleolytic activity is not the feature of Rat1 that ultimately promotes termination. Degradation of the nascent transcript allows Rat1 to pursue Pol II in a guided fashion and arrive at the site of RNA exit from Pol II. Upon this arrival, however, it is perhaps the specific and direct contact between Rat1 and Pol II that transmits the signal to terminate transcription.

H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.

We have earlier demonstrated the involvement of Mediator subunit Srb5/Med18 in the termination of transcription for a subset of genes in yeast. Srb5/Med18 could affect termination either indirectly by modulating CTD-Ser(2) phosphorylation near the 3' end of a gene or directly by physically interacting with the cleavage and polyadenylation factor or cleavage factor 1 (CF1) complex and facilitating their recruitment to the terminator region. Here, we show that the CTD-Ser(2) phosphorylation pattern on Srb5/Med18-dependent genes remains unchanged in the absence of Srb5 in cells. Coimmunoprecipitation analysis revealed the physical interaction of Srb5/Med18 with the CF1 complex. No such interaction of Srb5/Med18 with the cleavage and polyadenylation factor complex, however, could be detected. The Srb5/Med18-CF1 interaction was not observed in the looping defective sua7-1 strain. Srb5/Med18 cross-linking to the 3' end of genes was also abolished in the sua7-1 strain. Chromosome conformation capture analysis revealed that the looped architecture of Srb5/Med18-dependent genes was abrogated in srb5(-) cells. Furthermore, Srb5-dependent termination of transcription was compromised in the looping defective sua7-1 cells. The overall conclusion of these results is that gene looping plays a crucial role in Srb5/Med18 facilitated termination of transcription, and the looped gene architecture may have a general role in termination of transcription in budding yeast.

Urease is an essential component of gastric acid acclimation by Helicobacter pylori. The increased level of urease in gastric acidity is due, in part, to acid activation of the two-component system consisting of the membrane sensor HP0165 (ArsS) and its response regulator HP0166 (ArsR), which regulates transcription of the seven genes in two separate operons (ureAB and ureIEFGH) of the urease gene cluster. Recently, we identified a novel cis-encoded antisense small RNA, 5'ureB-sRNA, targeted at the 5' end of ureB, which downregulates ureAB expression by truncation of the ureAB transcript at neutral pH. It is not known whether the truncated transcript is due to transcription termination or processing of the full-length mRNA by codegradation of a ureAB mRNA-sRNA hybrid complex. S1 nuclease mapping assays show that the truncated transcript is due to transcription termination. Further studies using an in vitro transcription assay found that 5'ureB-sRNA promotes premature termination of transcription of ureAB mRNA. These results suggest that the antisense small RNA 5'ureB-sRNA downregulates ureAB expression by enhancing transcription termination 5' of ureB. With this mechanism, a limited amount of 5'ureB-sRNA is sufficient to regulate the relatively high level of ureAB transcript.

Two pathways of transcription termination, factor-independent and -dependent, exist in bacteria. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. NusG and NusA are also involved in antitermination of transcription at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes. We have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in the occurrence of RNA-DNA hybrids (R-loops), including from antisense and read-through transcripts, in a nusG missense mutant defective for Rho-dependent termination. Lethality associated with complete deficiency of Rho and NusG (but not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW, especially so on defined growth media. Our results suggest that factor-dependent transcription termination subserves a surveillance function to prevent translation-uncoupled transcription from generating R-loops, which would block replication fork progression and therefore be lethal, and that NusA performs additional essential functions as well in E. coli. Prevention of R-loop-mediated transcription-replication conflicts by cotranscriptional protein engagement of nascent RNA is emerging as a unifying theme among both prokaryotes and eukaryotes.

Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3' end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Transcription termination of RNA polymerase II between closely spaced genes is an important, though poorly understood, mechanism. This is true, in particular, in the Drosophila genome, where approximately 52% of tandem genes are separated by less than 1 kb. We show that a set of Drosophila tandem genes has a negative correlation of gene expression and display several molecular marks indicative of promoter pausing. We find that an intergenic spacing of 168 bp is sufficient for efficient transcription termination between the polo-snap tandem gene pair, by a mechanism that is independent of Pcf11 and Xrn2. In contrast, analysis of a tandem gene pair containing a longer intergenic region reveals that termination occurs farther downstream of the poly(A) signal and is, in this case, dependent on Pcf11 and Xrn2. For polo-snap, displacement of poised polymerase from the snap promoter by depletion of the initiation factor TFIIB results in an increase of polo transcriptional read-through. This suggests that poised polymerase is necessary for transcription termination. Interestingly, we observe that polo forms a TFIIB dependent gene loop between its promoter and terminator regions. Furthermore, in a plasmid containing the polo-snap locus, deletion of the polo promoter causes an increase in snap expression, as does deletion of polo poly(A) signals. Taken together, our results indicate that polo forms a gene loop and polo transcription termination occurs by an Xrn2 and Pcf11 independent mechanism that requires TFIIB.