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Marc Boudvillain - One of the best experts on this subject based on the ideXlab platform.
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A simple fluorescence microplate assay to monitor RNA-DNA hybrid unwinding by the bacterial transcription Termination Factor Rho
2021Co-Authors: Isabelle Simon, Marc BoudvillainAbstract: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.
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regulatory interplay between small rnas and transcription Termination Factor rho
Biochimica et Biophysica Acta, 2020Co-Authors: Lionello Bossi, Philippe Bouloc, Nara Figueroabossi, Marc BoudvillainAbstract:Abstract The largest and best studied group of regulatory small RNAs (sRNAs) in bacteria act by modulating translation or turnover of messenger RNAs (mRNAs) through base-pairing interactions that typically take place near the 5′ end of the mRNA. This allows the sRNA to bind the complementary target sequence while the remainder of the mRNA is still being made, creating conditions whereby the action of the sRNA can extend to transcriptional steps, most notably transcription Termination. Increasing evidence corroborates the existence of a functional interplay between sRNAs and Termination Factor Rho. Two general mechanisms have emerged. One mechanism operates in translated regions subjected to sRNA repression. By inhibiting ribosome binding co-transcriptionally, the sRNA uncouples translation from transcription, allowing Rho to bind the nascent RNA and promote Termination. In the second mechanism, which functions in 5′ untranslated regions, the sRNA antagonizes Termination directly by interfering with Rho binding to the RNA or the subsequent translocation along the RNA. Here, we review the above literature in the context of other mechanisms that underlie the participation of Rho-dependent transcription Termination in gene regulation. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.
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evaluating the effect of small rnas and associated chaperones on rho dependent Termination of transcription in vitro
Methods of Molecular Biology, 2018Co-Authors: Cedric Nadiras, Annie Schwartz, Mildred Delaleau, Marc BoudvillainAbstract:: Besides their well-known posttranscriptional effects on mRNA translation and decay, sRNAs and associated RNA chaperones (e.g., Hfq, CsrA) sometimes regulate gene expression at the transcriptional level. In this case, the sRNA-dependent machinery modulates the activity of the transcription Termination Factor Rho, a ring-shaped RNA translocase/helicase that dissociates transcription elongation complexes at specific loci of the bacterial genome. Here, we describe biochemical assays to detect Rho-dependent Termination signals in genomic regions of interest and to assess the effects of sRNAs and/or associated RNA chaperones on such signals.
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direct observation of the translocation mechanism of transcription Termination Factor rho
Nucleic Acids Research, 2015Co-Authors: Marc Boudvillain, Marcelo Nollmann, Emmanuel Margeat, Veronika Gocheva, Antoine Le GallAbstract:Rho is a ring-shaped, ATP-fueled motor essential for remodeling transcriptional complexes and R-loops in bacteria. Despite years of research on this fundamental model helicase, key aspects of its mechanism of translocation remain largely unknown. Here, we used single-molecule manipulation and fluorescence methods to directly monitor the dynamics of RNA translocation by Rho. We show that the efficiency of Rho activation is strongly dependent on the force applied on the RNA but that, once active, Rho is able to translocate against a large opposing force (at least 7 pN) by a mechanism involving ‘tethered tracking’. Importantly, the ability to directly measure dynamics at the single-molecule level allowed us to determine essential motor properties of Rho. Hence, Rho translocates at a rate of ∼56 nt per second under our experimental conditions, which is 2–5 times faster than velocities measured for RNA polymerase under similar conditions. Moreover, the processivity of Rho (∼62 nt at a 7 pN opposing force) is large enough for Rho to reach Termination sites without dissociating from its RNA loading site, potentially increasing the efficiency of transcription Termination. Our findings unambiguously establish ‘tethered tracking’ as the main pathway for Rho translocation, support ‘kinetic coupling’ between Rho and RNA polymerase during Rho-dependent Termination, and suggest that forces applied on the nascent RNA transcript by cellular substructures could have important implications for the regulation of transcription and its coupling to translation in vivo.
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Monitoring RNA unwinding by the transcription Termination Factor Rho from Mycobacterium tuberculosis.
Methods in Molecular Biology, 2014Co-Authors: François D’heygère, Annie Schwartz, Franck Coste, Bertrand Castaing, Marc BoudvillainAbstract:Transcription Termination Factor Rho is a ring-shaped, homo-hexamieric RNA translocase that dissociates transcription elongation complexes and transcriptional RNA-DNA duplexes (R-loops) in bacteria. The molecular mechanisms underlying these biological functions have been essentially studied with Rho enzymes from Escherichia coli or close Gram-negative relatives. However, phylo-divergent Rho Factors may have distinct properties. Here, we describe methods for the preparation and in vitro characterization (ATPase and helicase activities) of the Rho Factor from Mycobacterium tuberculosis, a specimen with uncharacteristic molecular and enzymatic features. These methods set the stage for future studies aimed at better defining the diversity of enzymatic properties of Rho across the bacterial kingdom.
Claes M Gustafsson - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial transcription Termination Factor 1 directs polar replication fork pausing
Nucleic Acids Research, 2016Co-Authors: Yonghong Shi, Viktor Posse, Xuefeng Zhu, Anne K Hyvarinen, Howard T Jacobs, Maria Falkenberg, Claes M GustafssonAbstract:During replication of nuclear ribosomal DNA (rDNA), clashes with the transcription apparatus can cause replication fork collapse and genomic instability. To avoid this problem, a replication fork barrier protein is situated downstream of rDNA, there preventing replication in the direction opposite rDNA transcription. A potential candidate for a similar function in mitochondria is the mitochondrial transcription Termination Factor 1 (MTERF1, also denoted mTERF), which binds to a sequence just downstream of the ribosomal transcription unit. Previous studies have shown that MTERF1 prevents antisense transcription over the ribosomal RNA genes, a process which we here show to be independent of the transcription elongation Factor TEFM. Importantly, we now demonstrate that MTERF1 arrests mitochondrial DNA (mtDNA) replication with distinct polarity. The effect is explained by the ability of MTERF1 to act as a directional contrahelicase, blocking mtDNA unwinding by the mitochondrial helicase TWINKLE. This conclusion is also supported by in vivo evidence that MTERF1 stimulates TWINKLE pausing. We conclude that MTERF1 can direct polar replication fork arrest in mammalian mitochondria.
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structure of mitochondrial transcription Termination Factor 3 reveals a novel nucleic acid binding domain
Biochemical and Biophysical Research Communications, 2010Co-Authors: Henrik Spahr, Tore Samuelsson, Martin B Hallberg, Claes M GustafssonAbstract:In mammalian cells, a family of mitochondrial transcription Termination Factors (MTERFs) regulates mitochondrial gene expression. MTERF family members share a ∼270 residues long MTERF-domain required for DNA binding and transcription regulation. However, the structure of this widely conserved domain is unknown. Here, we show that the MTERF-domain of human MTERF3 forms a half-doughnut-shaped right-handed superhelix. The superhelix is built from α-helical tandem repeats that display a novel triangular three-helix motif. This repeat motif, which we denote the MTERF-motif, is a conserved structural element present in proteins from metazoans, plants, and protozoans. Furthermore, a narrow, strongly positively charged nucleic acid-binding path is found in the middle of the concave side of the half-doughnut. This arrangement suggests a half clamp nucleic acid-binding mode for MTERF-domains.
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a family of putative transcription Termination Factors shared amongst metazoans and plants
Current Genetics, 2005Co-Authors: Tomas Linder, Tore Samuelsson, Claes M Gustafsson, Maria Falkenberg, Jordi Asincayuela, Chan Bae Park, Mina Pellegrini, Nilsgoran LarssonAbstract:The human mitochondrial transcription Termination Factor (mTERF) is involved in the regulation of transcription of the mitochondrial genome. Similarity searches and phylogenetic analysis demonstrate that mTERF is a member of large and complex protein family (the MTERF family) shared amongst metazoans and plants. Interestingly, we identify three novel MTERF genes in vertebrates, which all encode proteins with predicted mitochondrial localization. Members of the MTERF family have so far not been detected in fungi, supporting the notion that mitochondrial transcription regulation may have evolved separately in yeast and animal cells.
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The human mitochondrial transcription Termination Factor (mTERF) is fully active in vitro in the non-phosphorylated form
Journal of Biological Chemistry, 2005Co-Authors: Jordi Asin-cayuela, Thomas Schwend, Géraldine Farge, Claes M GustafssonAbstract:Abstract The human mitochondrial transcription Termination Factor (mTERF) is a 39-kDa protein that terminates transcription at the 3′-end of the 16 S rRNA gene and thereby controls expression of the ribosomal transcription unit of mitochondrial DNA. The transcription Termination activity of human mTERF has been notoriously difficult to study in vitro, and it has been suggested that the activity of the protein is regulated by posttranslational modifications or by protein polymerization. We here characterize the activity of recombinant human mTERF expressed in insect cells. We observed that mTERF efficiently promotes sequence-specific Termination in a completely recombinant and highly purified in vitro system for mitochondrial transcription. The Termination activity has a distinct polarity, and we observed complete transcription Termination when the mTERF-binding site is oriented in a forward position relative the heavy strand promoter but only partial transcription Termination when the binding site is in the reverse position. We analyzed the biochemical characteristics of the active mTERF protein and found that it is a stable monomer at physiological salt concentration. Structural analysis, including phosphostaining, two-dimensional electrophoresis, and electrospray mass spectrometry, detected no evidence of phosphorylation. We conclude that the monomeric human mTERF is fully active in its non-phosphorylated form and that the protein does not require additional cellular Factors to terminate mitochondrial transcription in vitro.
Hannah E Mischo - One of the best experts on this subject based on the ideXlab platform.
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cell cycle modulation of transcription Termination Factor sen1
Molecular Cell, 2018Co-Authors: Hannah E Mischo, Yujin Chun, Kevin M Harlen, Brendan M Smalec, Somdutta Dhir, Stirling L Churchman, Stephen BuratowskiAbstract:Summary Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased Termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other Termination Factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA Termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant Termination. We suggest that ncRNA levels and coding gene transcription Termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.
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cell cycle modulation of transcription Termination Factor sen1
Molecular Cell, 2018Co-Authors: Hannah E Mischo, Yujin Chun, Kevin M Harlen, Brendan M Smalec, Somdutta Dhir, Stirling L Churchman, Stephen BuratowskiAbstract:Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased Termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other Termination Factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA Termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant Termination. We suggest that ncRNA levels and coding gene transcription Termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.
Stephen Buratowski - One of the best experts on this subject based on the ideXlab platform.
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cell cycle modulation of transcription Termination Factor sen1
Molecular Cell, 2018Co-Authors: Hannah E Mischo, Yujin Chun, Kevin M Harlen, Brendan M Smalec, Somdutta Dhir, Stirling L Churchman, Stephen BuratowskiAbstract:Summary Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased Termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other Termination Factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA Termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant Termination. We suggest that ncRNA levels and coding gene transcription Termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.
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cell cycle modulation of transcription Termination Factor sen1
Molecular Cell, 2018Co-Authors: Hannah E Mischo, Yujin Chun, Kevin M Harlen, Brendan M Smalec, Somdutta Dhir, Stirling L Churchman, Stephen BuratowskiAbstract:Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased Termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other Termination Factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA Termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant Termination. We suggest that ncRNA levels and coding gene transcription Termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.
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yeast swd2 is essential because of antagonism between set1 histone methyltransferase complex and apt associated with pta1 Termination Factor
Journal of Biological Chemistry, 2012Co-Authors: Luis M Soares, Stephen BuratowskiAbstract:The Set1 complex (also known as complex associated with Set1 or COMPASS) methylates histone H3 on lysine 4, with different levels of methylation affecting transcription by recruiting various Factors to distinct regions of active genes. Neither Set1 nor its associated proteins are essential for viability with the notable exception of Swd2, a WD repeat protein that is also a subunit of the essential transcription Termination Factor APT (associated with Pta1). Cells lacking Set1 lose COMPASS recruitment but show increased promoter cross-linking of TFIIE large subunit and the serine 5 phosphorylated form of the Rpb1 C-terminal domain. Although Swd2 is normally required for bringing APT to genes, deletion of SET1 restores both viability and APT recruitment to a strain lacking Swd2. We propose a model in which Swd2 is required for APT to overcome antagonism by COMPASS.
Ingrid Grummt - One of the best experts on this subject based on the ideXlab platform.
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Termination of mammalian rdna replication polar arrest of replication fork movement by transcription Termination Factor ttf i
Cell, 1997Co-Authors: Josefkarl Gerber, Ingrid Grummt, Eric Gogel, Christian Berger, Michael Wallisch, Friedemann Muller, Friedrich GrummtAbstract:Abstract A replication fork barrier (RFB) at the 3′ end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3′ terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I–specific transcription Termination Factor TTF-I, or deletion of the Termination domain of TTF-I abolishes RFB activity. Thus, the same Factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.
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rna polymerase i transcription on nucleosomal templates the transcription Termination Factor ttf i induces chromatin remodeling and relieves transcriptional repression
The EMBO Journal, 1997Co-Authors: Gernot Langst, Thiemo A Blank, Peter B Becker, Ingrid GrummtAbstract:Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription Termination Factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription Factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a Termination Factor and a chromatin-specific transcription activator.
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DIFFERENT DOMAINS OF THE MURINE RNA POLYMERASE I-SPECIFIC Termination Factor MTTF-I SERVE DISTINCT FUNCTIONS IN TRANSCRIPTION Termination
The EMBO Journal, 1995Co-Authors: Raymond Evers, Udo Rudloff, Amke Smid, Friedrich Lottspeich, Ingrid GrummtAbstract:Abstract Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription Termination Factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription Termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription Factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.