Search

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs., Premature termination codons can cause early translation termination and lead to disease. Here the authors perform a screen to identify compounds with readthrough activity and show that these reduce eRF1 levels to suppress premature termination associated with cystic fibrosis.

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.

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 erh1Δ is achieved through precocious 3′-processing/termination of prt lncRNA synthesis, to wit: (i) erh1Δ does not affect the activity of the prt or pho1 promoters per se; (ii) de-repression by erh1Δ 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) erh1Δ 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 mmi1Δ 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, erh1Δ de-repression of pho1 was eliminated by mutating the Mmi1-binding sites in the prt lncRNA.

Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3′ end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11; the latter protein binds to RNA and the RNA polymerase II carboxy-terminal domain. Here, we used siRNA mediated knockdown and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3′ untranslated regions (3′-UTRs), we observed shifts from promoter proximal regions to the 3′-UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells cosedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis–trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3′ processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3′ processing/termination that acts via Ssu72.

How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process.

The system of long noncoding RNA (lncRNA)–mediated transcriptional interference that represses fission yeast phosphate homoeostasis gene pho1 provides a sensitive readout of genetic influences on cotranscriptional 3′-processing and termination and a tool for discovery of regulators of this phase of the Pol2 transcription cycle. Here, we conducted a genetic screen for relief of transcriptional interference that unveiled a mechanism by which Pol2 termination is enhanced via a gain-of-function mutation, G476S, in the RNA-binding domain of an essential termination factor, Seb1. The genetic and physical evidence for gain-of-function is compelling: 1) seb1-G476S de-represses pho1 and tgp1, both of which are subject to lncRNA-mediated transcriptional interference; 2) seb1-G476S elicits precocious lncRNA transcription termination in response to lncRNA 5′-proximal poly(A) signals; 3) seb1-G476S derepression of pho1 is effaced by loss-of-function mutations in cleavage and polyadenylation factor (CPF) subunits and termination factor Rhn1; 4) synthetic lethality of seb1-G476S with pho1 derepressive mutants rpb1-CTD-S7A and aps1∆ is rescued by CPF/Rhn1 loss-of-function alleles; and 5) seb1-G476S elicits an upstream shift in poly(A) site preference in several messenger RNA genes. A crystal structure of the Seb1-G476S RNA-binding domain indicates potential for gain of contacts from Ser476 to RNA nucleobases. To our knowledge, this is a unique instance of a gain-of-function phenotype in a eukaryal transcription termination protein.

Mitochondria are energy producing organelles of the eukaryotic cell, involved in the synthesis of key metabolites, calcium homeostasis and apoptosis. Protein biosynthesis in these organelles is a relic of its endosymbiotic origin. While mitochondrial translational factors have homologues among prokaryotes, they possess a number of unique traits. Remarkably as many as four mammalian mitochondrial proteins possess a clear similarity with translation termination factors. The review focuses on the ICT1, which combines several functions. It is a non-canonical termination factor for protein biosynthesis, a rescue factor for stalled mitochondrial ribosomes, a structural protein and a regulator of proliferation, cell cycle, and apoptosis. Such a diversity of roles demonstrates the high functionality of mitochondrial translation associated proteins and their relationship with numerous processes occurring in a living cell.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3’-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3’-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.

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.

SUMMARY Regulation of cleavage and polyadenylation (CPA) affects gene expression and polyadenylation site (PAS) choice. Here, we report that the CPA and termination factor PCF11 modulates gene expression on the basis of gene size. Although downregulation of PCF11 leads to inhibition of short gene expression, long genes are upregulated because of suppressed intronic polyadenylation (IPA) enriched in large introns. We show that this regulatory scheme, named PCF11-mediated expression regulation through IPA (PEIPA), takes place in cell differentiation, during which downregulation of PCF11 is coupled with upregulation of long genes with functions in cell morphology, adhesion, and migration. PEIPA targets distinct gene sets in different cell contexts with similar rules. Furthermore, PCF11 is autoregulated through a conserved IPA site, the removal of which leads to global activation of PASs close to gene promotors. Therefore, PCF11 uses distinct mechanisms to regulate genes of different sizes, and its autoregulation maintains homeostasis of PAS usage in the cell., In Brief Wang et al. report a gene size-based dichotomic scheme by which the cleavage and polyadenylation factor PCF11 modulates gene expression. Distinct polyA site types are involved to regulate short and long genes. PCF11 expression level is autoregulated and is connected with long gene expression in cell differentiation and across tissues., Graphical Abstract

Eukaryotic translation termination is mediated by two conserved interacting release factors, eRF1 and eRF3. eRF1 recognizes the stop codon and promotes the hydrolysis of the polypeptide chain, while eukaryotic eRF3 stimulates eRF1 release activity in the presence of GTP. It is widely believed that translation termination is highly conserved in eukaryotes. However, recent results that eRF1 is present in multiple, partially redundant copies in plants and that eRF1 expression is controlled by a complex, plant-specific autoregulatory circuit suggest that regulation of translation termination might be especially complex in plants. Surprisingly, very little is known about translation termination in plant, for instance, the eRF3 termination factor has not been analyzed in plants yet. Thus, we wanted to identify and characterize the eRF3 translation termination factor in plants. By combining a range of transient and transgenic assay here, we identified plant eRF3 and showed that it directly interacts with eRF1. In contrast to eRF1, plant eRF3 is not autoregulated, while eRF3 and eRF1 expressions are connected. We also demonstrated that eRF3 interacts with the core NMD factor, UPF1, and the expression of eRF3 is NMD regulated in certain plant species suggesting that in addition to the normal translation termination, eRF3 could be connected to plant nonsense-mediated decay (NMD). Finally, it appears that the plant termination factors are present in physiologically different concentrations, while eRF1 concentration limits the efficiency of both translation termination and NMD, eRF3 is present in non-limiting concentration.

International audience; Transcription termination factor Rho contributes to shape the transcriptomes of many bacteria and is essential in a large subset of them. Although the transcription termination function of Rho is not always easy to reconstitute and to study in vitro, assays based on the ATPdependent RNA-DNA hybrid unwinding activity of the factor can prove useful to dissect Rho mechanisms or to seek new antibiotics targeting Rho. However, current in vitro assays of Rho helicase activity are time-consuming, as they usually require radiolabeling of the hybrid substrates and analysis of reaction products by gel electrophoresis. Here, we describe a fluorescence-based microplate assay that informs on Rho helicase activity in a matter of minutes and allows the multiplexed analysis of conditions required for primary biochemical characterization or for drug screening.

A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences (“repeats”) interspersed with short, variable sequences (“spacers”). CRISPR arrays are transcribed and processed into individual CRISPR RNAs (crRNAs) that each include a single spacer, and direct Cas proteins to complementary sequence in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that Rho termination functionally limits the length of bacterial CRISPR arrays, and we identify a widespread antitermination mechanism that antagonizes Rho to facilitate complete transcription of CRISPR arrays. Thus, our data highlight the importance of Rho termination in the evolution of bacterial CRISPR-Cas systems.

Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs., Transcription in archaea is known to be regulated through the recruitment of RNA polymerase to promoters. Here, the authors show that the archaeon Saccharolobus solfataricus regulates transcription globally through a rate-limiting promoter-proximal elongation step.

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination., info:eu-repo/semantics/published

Nonsense mutations are single nucleotide variations within the coding sequence of a gene that result in a premature termination codon (PTC). The occurrence of such PTCs most often leads to a complete loss of protein function and a reduction in messenger ribonucleic acid (mRNA) levels due to the nonsense-mediated mRNA decay (NMD), a cellular surveillance mechanism that triggers selective degradation of mutant transcripts. Therapeutic approaches to circumvent the consequences of nonsense mutations may act at different levels: (i) the genomic deoxyribonucleic acid (DNA) by replacing the defective gene; (ii) the mRNA by inducing the excision of the mutation-bearing exon during splicing, or by inhibiting the NMD-associated degradation and (iii) the protein by suppressing the premature termination of translation using transfer RNA (tRNA) suppressors or drugs inducing readthrough. Indeed, a combination of these approaches may be necessary, and it is most likely that they will lead to a mutation-specific, personalised medicine. Key Concepts: Nonsense mutations may lead to loss or gain of function pathological mechanisms. Premature termination codons (PTC) resulting from nonsense mutations trigger transcript degradation through the nonsense-mediated mRNA decay (NMD) mechanism. Mutant mRNA escaping NMD lead to the synthesis of truncated proteins potentially deleterious. Skipping of nonsense mutation-bearing exons can be induced by antisense oligonucleotides and leads to internally deleted proteins that retain some functionality. tRNA suppressors enable the reintroduction of a ‘sense’ amino acid and the translation of the full-length protein by competing with the termination factor eRF1. Drug-induced readthrough of PTC may allow synthesis of full-length functional proteins. Synergetic action between NMD inhibitors and readthrough inducers may potentialise reexpression of full-length proteins with restored functionality. Therapeutic approaches will most likely be mutation and disease specific. Keywords: nonsense mutations; premature termination codons; inherited disorders; therapeutic approaches; exon skipping; antisense oligonucleotides; nonsense mediated decay; tRNA suppressors; readthrough; aminoglycosides

Diketopiperazines (DKPs) are a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp3 carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. By observing bicyclomycin production from heterologously expression of the pseudomonas bcm gene cluster, we establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

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.

9 p.-5 fig., Amyloid aggregation of the eukaryotic translation terminator eRF3/Sup35p, the [PSI+] prion, empowers yeast ribosomes to read-through UGA stop codons. No similar functional prion, skipping a stop codon, has been found in Escherichia coli, a fact possibly due to the efficient back-up systems found in bacteria to rescue non-stop complexes. Here we report that engineering hydrophobic amyloidogenic repeats from a synthetic bacterial prion-like protein (RepA-WH1) into the E. coli releasing factor RF1 promotes its aggregation and enables ribosomes to continue with translation through a premature UAG stop codon located in a β-galactosidase reporter. To our knowledge, intended aggregation of a termination factor is a way to overcome the bacterial translation quality checkpoint that had not been reported so far. We also show the feasibility of using the amyloidogenic RF1 chimeras as a reliable, rapid and cost-effective system to screen for molecules inhibiting intracellular protein amyloidogenesis in vivo, by testing the effect on the chimeras of natural polyphenols with known anti-amyloidogenic properties. Resveratrol exhibits a clear amyloid-solubilizing effect in this assay, showing no toxicity to bacteria or interference with the enzymatic activity of β-galactosidase., This work has been financed with grants (CSD2009-00088, BIO2012-30852 and BIO2015-68730-R) from Spanish AEI and UE-FEDER. R.G. is a member of the CIB-CSIC Intramural Research Program “Macromolecular Machines for Better Life” (MACBET).

Ribosomes that translate mRNAs lacking stop codons become stalled at the 3' end of the mRNA. Recycling of these stalled ribosomes is essential for cell viability. In bacteria three ribosome rescue systems have been identified so far, with the most ubiquitous and best characterized being the trans-translation system mediated by transfer-messenger RNA (tmRNA) and small protein B (SmpB). The two additional rescue systems present in some bacteria employ alternative rescue factor (Arf) A and release factor (RF) 2 or ArfB. Recent structures have revealed how ArfA mediates ribosome rescue by recruiting the canonical termination factor RF2 to ribosomes stalled on truncated mRNAs. This now provides us with the opportunity to compare and contrast the available structures of all three bacterial ribosome rescue systems.

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.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3' untranslated region (3'-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3'-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3'-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.

The superfamily 1B (SF1B) helicase Sen1 is an essential protein that plays a key role in the termination of non‐coding transcription in yeast. Here, we identified the ~90 kDa helicase core of Saccharomyces cerevisiae Sen1 as sufficient for transcription termination in vitro and determined the corresponding structure at 1.8 A resolution. In addition to the catalytic and auxiliary subdomains characteristic of the SF1B family, Sen1 has a distinct and evolutionarily conserved structural feature that “braces” the helicase core. Comparative structural analyses indicate that the “brace” is essential in shaping a favorable conformation for RNA binding and unwinding. We also show that subdomain 1C (the “prong”) is an essential element for 5′‐3′ unwinding and for Sen1‐mediated transcription termination in vitro . Finally, yeast Sen1 mutant proteins mimicking the disease forms of the human orthologue, senataxin, show lower capacity of RNA unwinding and impairment of transcription termination in vitro . The combined biochemical and structural data thus provide a molecular model for the specificity of Sen1 in transcription termination and more generally for the unwinding mechanism of 5′‐3′ helicases.

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.

Pervasive transcription is widespread and needs to be controlled in order to avoid interference with gene expression. In Saccharomyces cerevisiae, the highly conserved helicase Sen1 plays a key role in restricting pervasive transcription by eliciting early termination of non-coding transcription. However, many aspects of the mechanism of termination remain unclear. In this study we characterize the biochemical activities of Sen1 and their role in termination. First, we demonstrate that the helicase domain (HD) is sufficient to dissociate the elongation complex (EC) in vitro. Both full-length Sen1 and its HD can translocate along single-stranded RNA and DNA in the 5΄ to 3΄ direction. Surprisingly, however, we show that Sen1 is a relatively poorly processive enzyme, implying that it must be recruited in close proximity to the RNA polymerase II (RNAPII) for efficient termination. We present evidence that Sen1 can promote forward translocation of stalled polymerases by acting on the nascent transcript. In addition, we find that dissociation of the EC by Sen1 is favoured by the reannealing of the DNA upstream of RNAPII. Taken together, our results provide new clues to understand the mechanism of Sen1-dependent transcription termination and a rationale for the kinetic competition between elongation and termination.

The human DEAD-box RNA-helicase DDX19 functions in mRNA export through the nuclear pore complex. The yeast homolog of this protein, Dbp5, has been reported to participate in translation termination. Using a reconstituted mammalian in vitro translation system, we show that the human protein DDX19 is also important for translation termination. It is associated with the fraction of translating ribosomes. We show that DDX19 interacts with pre-termination complexes (preTCs) in a nucleotide-dependent manner. Furthermore, DDX19 increases the efficiency of termination complex (TC) formation and the peptide release in the presence of eukaryotic release factors. Using the eRF1(AGQ) mutant protein or a non-hydrolysable analog of GTP to inhibit subsequent peptidyl-tRNA hydrolysis, we reveal that the activation of translation termination by DDX19 occurs during the stop codon recognition. This activation is a result of DDX19 binding to preTC and a concomitant stabilization of terminating ribosomes. Moreover, we show that DDX19 stabilizes ribosome complexes with translation elongation factors eEF1 and eEF2. Taken together, our findings reveal that the human RNA helicase DDX19 actively participates in protein biosynthesis.

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). Using disruption-compensation (DisCo) protein-protein interaction network analysis, interaction changes were observed within the termination machinery as a consequence of deletion of the serine 5 RNAPII CTD phosphatase Rtr1. 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. These changes could be the result of altered interactions between the termination machinery and/or increased levels of premature termination of RNAPII. Transcriptome analysis in rtr1Δ cells found decreased pervasive transcription and a shift in balance of expression of sense and antisense transcripts. Globally, rtr1Δ leads to decreases in 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 and increased efficiency of snRNA termination. Additionally, premature termination increases globally at protein-coding genes where NNS is recruited during early elongation. The effects of rtr1Δ on RNA expression levels were erased following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade terminated noncoding RNAs. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature RNAPII termination of mRNAs and ncRNAs. Additionally, Rtr1 phosphatase activity facilitates low-level elongation of noncoding transcripts that impact the transcriptome through RNAPII interference.AUTHOR SUMMARYMany 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. Additionally, many of the prematurely terminated noncoding RNA transcripts are degraded by the Rrp6-containing nuclear exosome, a known component of the Nrd1-Nab3 termination coupled RNA degradation pathway. These findings suggest that Rtr1 normally promotes elongation of RNA Polymerase II transcripts through preventation of Nrd1-directed termination.

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.

The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(l-isoleucyl-l-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.

Ribonucleoprotein (RNP) granules are higher order assemblies of RNA, RNA-binding proteins, and other proteins, that regulate the transcriptome and protect RNAs from environmental challenge. There is a diverse range of RNP granules, many cytoplasmic, which provide various levels of regulation of RNA metabolism. Here we present evidence that the yeast transcription termination factor, Nab3, is targeted to intranuclear granules in response to glucose starvation by Nab3's proline/glutamine-rich, prion-like domain (PrLD) which can assemble into amyloid in vitro. Localization to the granule is reversible and sensitive to the chemical probe 1,6 hexanediol suggesting condensation is driven by phase separation. Nab3's RNA recognition motif is also required for localization as seen for other PrLD-containing RNA-binding proteins that phase separate. Although the PrLD is necessary, it is not sufficient to localize to the granule. A heterologous PrLD that functionally replaces Nab3's essential PrLD, directed localization to the nuclear granule, however a chimeric Nab3 molecule with a heterologous PrLD that cannot restore termination function or viability, does not form granules. The Nab3 nuclear granule shows properties similar to well characterized cytoplasmic compartments formed by phase separation, suggesting that, as seen for other elements of the transcription machinery, termination factor condensation is functionally important.

The carboxy-terminal domain (CTD) code encrypted within the Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats of RNA polymerase II (Pol2) is deeply rooted in eukaryal biology. Key steps to deciphering the code are identifying the events in gene expression that are governed by individual “letters” and then defining a vocabulary of multiletter “words” and their meaning. Thr4 and Ser7 exert opposite effects on the fission yeast pho1 gene, expression of which is repressed under phosphate-replete conditions by transcription of an upstream flanking long noncoding RNA (lncRNA). Here we attribute the derepression of pho1 by a CTD-S7A mutation to precocious termination of lncRNA synthesis, an effect that is erased by mutations of cleavage-polyadenylation factor (CPF) subunits Ctf1, Ssu72, Ppn1, Swd22, and Dis2 and termination factor Rhn1. By contrast, a CTD-T4A mutation hyperrepresses pho1, as do CPF subunit and Rhn1 mutations, implying that T4A reduces lncRNA termination. Moreover, CTD-T4A is synthetically lethal with ppn1∆ and swd22∆, signifying that Thr4 and the Ppn1•Swd22 module play important, functionally redundant roles in promoting Pol2 termination. We find that Ppn1 and Swd22 become essential for viability when the CTD array is curtailed and that S7A overcomes the need for Ppn1•Swd22 in the short CTD context. Mutational synergies highlight redundant essential functions of (i) Ppn1•Swd22 and Rhn1, (ii) Ppn1•Swd22 and Ctf1, and (iii) Ssu72 and Dis2 phosphatases. CTD alleles Y1F, S2A, and T4A have overlapping synthetic lethalities with ppn1∆ and swd22∆, suggesting that Tyr1-Ser2-Thr4 form a three-letter CTD word that abets termination, with Rhn1 being a likely “reader” of this word.

The [PSI+] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI+] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI+], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI+] formation, while Sla2 can prevent [PSI+] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI+] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI+] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI+] detoxification system.

The [PSI+] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI+] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI+], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI+] formation, while Sla2 can prevent [PSI+] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI+] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI+] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI+] detoxification system.

Transcription Timely and tunable cessation of RNA synthesis is vital for cellular homeostasis. RNA helicases such as the archetypal termination factor r actively dismantle transcription complexes, but the transitory nature of termination makes the process hard to study structurally. Said et al. assembled ρ-bound transcription complexes and studied them using cryo–electron microscopy with an approach that captured a series of functional states en route to termination. They found an extensive and dynamic network of r interactions with RNA polymerase, nucleic acids, and accessory Nus factors. ρ mediates stepwise rearrangements of these contacts, transforming an actively transcribing complex into a moribund pretermination intermediate. Science , this issue p. [eabd1673][1] [1]: /lookup/doi/10.1126/science.abd1673

Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3′ end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1–Nab3–Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3′ end processing factors, is enriched at the 3′ end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3′ untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3′ end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation.

TT-Seq maps a transient transcriptome RNA expression is related to protein abundance and cellular function. However, the amounts of RNA generated at any one time-point have been difficult to determine. Schwalb et al. developed a method, transient transcriptome sequencing (TT-Seq), to collect and sequence all RNA segments synthesized over 5 minutes. Because 5 minutes is not long enough to fully degrade even the most transient RNA, this method can detect the synthesis of most RNA without bias. Applying this method to human K562 cells, TT-Seq detected thousands of noncoding transcripts, providing a snapshot of RNA synthesis rates and RNA half-lives, and full-length maps of short-lived RNAs such as enhancers and short intergenic noncoding RNAs. Science , this issue p. 1225

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.