M. S. Ciampi, Rho-dependent terminators and transcription termination, Microbiology, vol.152, pp.2515-2528, 2006.

M. Boudvillain, N. Figueroa-bossi, and L. Bossi, Terminator still moving forward: expanding roles for Rho factor, Curr. Opin. Microbiol, vol.16, pp.118-124, 2013.
URL : https://hal.archives-ouvertes.fr/hal-00840158

A. Ray-soni, M. J. Bellecourt, and R. Landick, Mechanisms of bacterial transcription Termination: All good things must end, Annu. Rev. Biochem, vol.85, pp.319-347, 2016.

E. Nudler, RNA polymerase backtracking in gene regulation and genome instability, Cell, vol.149, pp.1438-1445, 2012.

R. S. Washburn and M. E. Gottesman, Regulation of transcription elongation and termination, Biomolecules, vol.5, pp.1063-1078, 2015.

A. Grylak-mielnicka, V. Bidnenko, J. Bardowski, and E. Bidnenko, Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria, Microbiology, vol.162, pp.433-447, 2016.

L. V. Richardson and J. P. Richardson, Rho-dependent termination of transcription is governed primarily by the upstream Rho utilization (rut) sequences of a terminator, J. Biol. Chem, vol.271, pp.21597-21603, 1996.

M. Guerin, N. Robichon, J. Geiselmann, and A. R. Rahmouni, A simple polypyrimidine repeat acts as an artificial Rho-dependent terminator in vivo and in vitro, Nucleic Acids Res, vol.26, pp.4895-4900, 1998.
URL : https://hal.archives-ouvertes.fr/hal-02127232

K. Hollands, S. Proshkin, S. Sklyarova, V. Epshtein, A. Mironov et al., Riboswitch control of Rho-dependent transcription termination, Proc. Natl. Acad. Sci. U.S.A, vol.109, pp.5376-5381, 2012.

N. Figueroa-bossi, A. Schwartz, B. Guillemardet, F. D'heygere, L. Bossi et al., RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination, Genes Dev, vol.28, pp.1239-1251, 2014.
DOI : 10.1101/gad.240192.114
URL : https://hal.archives-ouvertes.fr/hal-01178971

M. A. Kriner, A. Sevostyanova, and E. A. Groisman, Learning from the Leaders: Gene regulation by the transcription termination factor rho, Trends Biochem. Sci, vol.41, pp.690-699, 2016.

M. Rabhi, O. Espeli, A. Schwartz, B. Cayrol, A. R. Rahmouni et al., The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators, EMBO J, vol.30, pp.2805-2816, 2011.
URL : https://hal.archives-ouvertes.fr/hal-00720713

P. Alifano, F. Rivellini, D. Limauro, C. B. Bruni, and M. S. Carlomagno, A consensus motif common to all Rho-dependent prokaryotic transcription terminators, Cell, vol.64, pp.553-563, 1991.

M. Rabhi, A. R. Rahmouni, and M. Boudvillain, RNA Helicases, vol.19, pp.243-271, 2010.
URL : https://hal.archives-ouvertes.fr/hal-00602496

C. E. Bogden, D. Fass, N. Bergman, M. D. Nichols, and J. M. Berger, The structural basis for terminator recognition by the Rho transcription termination factor, Mol Cell, vol.3, pp.487-493, 1999.

E. Skordalakes and J. M. Berger, Structure of the Rho transcription termination factor Rho from Escherichia coli, Methods Mol. Biol, vol.587, pp.137-154, 2003.

M. Rabhi, V. Gocheva, F. Jacquinot, A. Lee, E. Margeat et al., Mutagenesis-based evidence for an asymmetric configuration of the ring-shaped transcription termination factor Rho, J. Mol. Biol, vol.405, pp.497-518, 2011.
URL : https://hal.archives-ouvertes.fr/hal-00608241

I. Artsimovitch and T. M. Henkin, In vitro approaches to analysis of transcription termination, Methods, vol.47, pp.37-43, 2009.

M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res, vol.31, pp.3406-3415, 2003.
DOI : 10.1093/nar/gkg595
URL : https://academic.oup.com/nar/article-pdf/31/13/3406/9487491/gkg595.pdf

E. Lennart, T. Johan, and W. Svante, CV-ANOVA for significance testing of PLS and OPLS® models, J. Chemometrics, vol.22, pp.594-600, 2008.

M. N. Triba, L. Le-moyec, R. Amathieu, C. Goossens, N. Bouchemal et al., , 2015.

, PLS/OPLS models in metabolomics: the impact of permutation of dataset rows on the K-fold cross-validation quality parameters, Mol. bioSyst, vol.11, pp.13-19

A. Medina-rivera, M. Defrance, O. Sand, C. Herrmann, J. A. Castro-mondragon et al., RSAT 2015: Regulatory sequence analysis tools, vol.43, pp.50-56, 2015.
DOI : 10.1093/nar/gkv362
URL : https://hal.archives-ouvertes.fr/hal-01624369

E. Skordalakes and J. M. Berger, Structural Insights into RNA-Dependent ring closure and ATPase activation by the rho termination factor, Cell, vol.127, pp.553-564, 2006.

H. Abdi and L. J. Williams, Principal component analysis, Comput. Stat, vol.2, pp.433-459, 2010.
URL : https://hal.archives-ouvertes.fr/hal-01259094

M. Bylesjöbylesj¨bylesjö, M. Rantalainen, O. Cloarec, J. K. Nicholson, E. Holmes et al., OPLS discriminant analysis: combining the strengths of PLS-DA and SIMCA classification, J. Chemometrics, vol.20, pp.341-351, 2006.

S. Gama-castro, H. Salgado, A. Santos-zavaleta, D. Ledezma-tejeida, L. Muniz-rascado et al., RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond, Nucleic Acids Res, vol.44, pp.133-143, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01460125

G. Kudla, A. W. Murray, D. Tollervey, and J. B. Plotkin, Coding-sequence determinants of gene expression in Escherichia coli, Science, vol.324, pp.255-258, 2009.

J. P. Richardson, Rho-dependent transcription termination, Biochim. Biophys. Acta, vol.1048, pp.127-138, 1990.
DOI : 10.1016/0167-4781(90)90048-7

W. D. Morgan, D. G. Bear, and P. H. Hippel, Rho-dependent termination of transcription. I. Identification and characterization of termination sites for transcription from the bacteriophage lambda PR promoter, J. Biol. Chem, vol.258, pp.9553-9564, 1983.

A. Q. Zhu and P. H. Hippel, Rho-dependent termination within the trp t' terminator. I. Effects of rho loading and template sequence, Biochemistry, vol.37, pp.11202-11214, 1998.

M. R. Lawson, K. Dyer, and J. M. Berger, Ligand-induced and small-molecule control of substrate loading in a hexameric helicase, Proc. Natl. Acad. Sci. U.S.A, vol.113, pp.13714-13719, 2016.

I. O. Vvedenskaya, H. Vahedian-movahed, J. G. Bird, J. G. Knoblauch, S. R. Goldman et al., Interactions between RNA polymerase and the "core recognition element" counteract pausing, Science, vol.344, pp.1285-1289, 2014.
DOI : 10.1126/science.1253458
URL : http://europepmc.org/articles/pmc4277259?pdf=render

M. H. Larson, R. A. Mooney, J. M. Peters, T. Windgassen, D. Nayak et al., A pause sequence enriched at translation start sites drives transcription dynamics in vivo, Science, vol.344, pp.1042-1047, 2014.

T. J. Santangelo and I. Artsimovitch, Termination and antitermination: RNA polymerase runs a stop sign, Nat. Rev. Microbiol, vol.9, pp.319-329, 2011.

J. K. Leela, A. H. Syeda, K. Anupama, and J. Gowrishankar, Rho-dependent transcription termination is essential to prevent excessive genome-wide R-loops in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A, vol.110, pp.258-263, 2013.

L. Bastet, A. Chauvier, N. Singh, A. Lussier, A. M. Lamontagne et al., Translational control and Rho-dependent transcription termination are intimately linked in riboswitch regulation, Nucleic Acids Res, vol.45, pp.7474-7486, 2017.

P. Alifano, F. Rivellini, D. Limauro, C. B. Bruni, and M. S. Carlomagno, A consensus motif common to all Rho-dependent prokaryotic transcription terminators, Cell, vol.64, pp.553-563, 1991.

L. Bossi, A. Schwartz, B. Guillemardet, M. Boudvillain, and N. Figueroa-bossi, A role for Rhodependent polarity in gene regulation by a noncoding small RNA, Genes Dev, vol.26, pp.1864-1873, 2012.
URL : https://hal.archives-ouvertes.fr/hal-00727260

R. Menouni, S. Champ, L. Espinosa, M. Boudvillain, and M. Ansaldi, Transcription termination controls prophage maintenance in Escherichia coli genomes, Proc Natl Acad Sci U S A, vol.110, pp.14414-14419, 2013.
URL : https://hal.archives-ouvertes.fr/hal-00874882

V. Stewart, R. Landick, and C. Yanofsky, Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12, J Bacteriol, vol.166, pp.217-223, 1986.

R. C. Wek, J. H. Sameshima, and G. W. Hatfield, Rho-dependent transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12, J Biol Chem, vol.262, pp.15256-15261, 1987.

E. C. Ruteshouser and J. P. Richardson, Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene, J Mol Biol, vol.208, pp.23-43, 1989.

N. Figueroa-bossi, A. Schwartz, B. Guillemardet, F. D'heygere, L. Bossi et al., RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination, Genes Dev, vol.28, pp.1239-1251, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01178971

H. Kupper, T. Sekiya, M. Rosenberg, J. Egan, and A. Landy, A rho-dependent termination site in the gene coding for tyrosine tRNA su3 of Escherichia coli, Nature, vol.272, pp.423-428, 1978.

F. Zalatan, J. Galloway-salvo, and T. Platt, Deletion analysis of the Escherichia coli rhodependent transcription terminator trp t, J. Biol. Chem, vol.268, pp.17051-17056, 1993.

W. D. Morgan, D. G. Bear, and P. H. Hippel, Rho-dependent termination of transcription. I. Identification and characterization of termination sites for transcription from the bacteriophage lambda PR promoter, J. Biol. Chem, vol.258, pp.9553-9564, 1983.

P. Alifano, F. Rivellini, D. Limauro, C. B. Bruni, and M. S. Carlomagno, A consensus motif common to all Rho-dependent prokaryotic transcription terminators, Cell, vol.64, pp.553-563, 1991.

K. Hollands, S. Proshkin, S. Sklyarova, V. Epshtein, A. Mironov et al., Riboswitch control of Rho-dependent transcription termination, Proc Natl Acad Sci U S A, vol.109, pp.5376-5381, 2012.

, 1× T 10 E 1 buffer: 10 mM Tris-HCl, 1 mM EDTA

, #N3233S ladder from New England Biolabs)

, 6× agarose gel loading buffer: 15% Ficoll-400, 0.1% SDS, 0.1% bromophenol blue, 20 mM Tris-HCl, 66 mM EDTA

, 20× TAE buffer: dissolve 98 g of Tris base in approximately 800 mL of water, then add 22.8 mL of glacial acetic acid and 40 mL of 0.5 M EDTA pH 8.0, and add, vol.1

, 1× TAE buffer: obtained by dilution of the 20× TAE stock buffer

, Microspin columns from GE Healthcare)

, UV spectrophotometer suitable for micro-volumes measurements, Nanodrop 2000c from Thermo Scientific)

, 5× Transcription buffer: 0.12 M MgCl 2 , 0.4 M HEPES, pH 7.5, 0.1 M DTT, 0.05% Triton X-100, and 5 mM Spermidine

U. ,

, U/?L RQ1 DNase (Promega)

, Deionized RNA-grade water (see Note 4)

, Set of high-grade rNTPs (100 mM each)

, 24:1) mix, pH 6, Phenol:Chloroform:Isoamyl alcohol, vol.25

, Microspin columns from GE Healthcare)

, Preparation of the Rho protein from E. coli is detailed in the, ?M Rho stock solution (see Note 6) in Rho storage buffer (50% glycerol, 100 mM KCl, 0.1 mM EDTA, 0.1 mM DTT, 10 mM Tris-HCl, pH 7.9, vol.587

, ?M NusG stock solution (see Note 8)

, 2 ?M stock solution of CsrA or Hfq chaperone (optional) (see Note 8)

E. U/?l and . Polymerase,

, Stocks should include the sRNAs under investigation (i.e., the ones expected to pair with the mRNA target) and at least one negative control (sRNA not expected to bind to the mRNA target). We also like to include a shorter, synthetic oligoribonucleotide that is fully complementary to the mRNA sequence targeted by the sRNA(s) and serves as positive control

, 5× transcription termination buffer: 250 mM KCl, 25 mM MgCl 2 , 7.5 mM DTT, 0.25 mg/mL bovine serum albumin, and 200 mM Tris-HCl

, 10× initiation mixture: 2 mM ATP, 2 mM GTP, 2 mM CTP, 0.2 mM UTP, 250 ?g/mL rifampicin, and 2 ?Ci/?L 32 P-?UTP in 1× transcription termination buffer

O. Porrua, M. Boudvillain, and D. Libri, Transcription termination: variations on common themes, Trends Genet, vol.32, pp.508-522, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01407669

A. Ray-soni, M. J. Bellecourt, and R. Landick, Mechanisms of bacterial transcription termination: all good things must end, Annu Rev Biochem, vol.85, pp.319-347, 2016.

T. J. Santangelo and I. Artsimovitch, Termination and antitermination: RNA polymerase runs a stop sign, Nat Rev Microbiol, vol.9, pp.319-329, 2011.

M. A. Kriner, A. Sevostyanova, and E. A. Groisman, Learning from the leaders: gene regulation by the transcription termination factor Rho, Trends Biochem Sci, vol.41, pp.690-699, 2016.

K. Gish and C. Yanofsky, Evidence suggesting cis action by the TnaC leader peptide in regulating transcription attenuation in the tryptophanase operon of Escherichia coli, J Bacteriol, vol.177, pp.7245-7254, 1995.

Y. Matsumoto, K. Shigesada, and M. Hirano, Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: localization and function of its attenuators, J Bacteriol, vol.166, pp.945-958, 1986.

H. Yakhnin, J. E. Babiarz, and A. V. Yakhnin, Expression of the Bacillus subtilis trpEDCFBA operon is influenced by translational coupling and Rho termination factor, J Bacteriol, vol.183, pp.5918-5926, 2001.

K. Hollands, S. Proshkin, and S. Sklyarova, Riboswitch control of Rho-dependent transcription termination, Proc Natl Acad Sci U S A, vol.109, pp.5376-5381, 2012.

N. Takemoto, Y. Tanaka, and M. Inui, Rho and RNase play a central role in FMN riboswitch regulation in Corynebacterium glutamicum, Nucleic Acids Res, vol.43, pp.520-529, 2015.

N. Sedlyarova, I. Shamovsky, and B. K. Bharati, sRNA-mediated control of transcription termination in E. coli, Cell, vol.167, pp.111-121, 2016.

N. Figueroa-bossi, A. Schwartz, and B. Guillemardet, RNA remodeling by bacterial global regulator CsrA promotes Rhodependent transcription termination, Genes Dev, vol.28, pp.1239-1251, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01178971

M. G. Jorgensen, M. K. Thomason, and J. Havelund, Dual function of the McaS small RNA in controlling biofilm formation, Genes Dev, vol.27, pp.1132-1145, 2013.

X. Wang, A. K. Dubey, and K. Suzuki, CsrA post-transcriptionally represses pgaABCD, responsible for synthesis of a biofilm polysaccharide adhesin of Escherichia coli, Mol Microbiol, vol.56, pp.1648-1663, 2005.

L. Bossi, A. Schwartz, and B. Guillemardet, A role for Rho-dependent polarity in gene regulation by a noncoding small RNA, Genes Dev, vol.26, pp.1864-1873, 2012.
URL : https://hal.archives-ouvertes.fr/hal-00727260

X. Wang, S. C. Ji, and H. J. Jeon, Two-level inhibition of galK expression by spot 42: degradation of mRNA mK2 and enhanced transcription termination before the galK gene, Proc Natl Acad Sci U S A, vol.112, pp.7581-7586, 2015.

A. V. Yakhnin and P. Babitzke, NusG/Spt5: are there common functions of this ubiquitous transcription elongation factor?, Curr Opin Microbiol, vol.18, pp.68-71, 2014.

M. Rabhi, O. Espeli, and A. Schwartz, The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators, EMBO J, vol.30, pp.2805-2816, 2011.
URL : https://hal.archives-ouvertes.fr/hal-00720713

J. M. Peters, R. A. Mooney, and J. A. Grass, Rho and NusG suppress pervasive antisense transcription in Escherichia coli, Genes Dev, vol.26, pp.2621-2633, 2012.

R. Shashni, M. Z. Qayyum, and V. Vishalini, Redundancy of primary RNAbinding functions of the bacterial transcription terminator Rho, Nucleic Acids Res, vol.42, pp.9677-9690, 2014.

M. Boudvillain, C. Walmacq, and A. Schwartz, Simple enzymatic assays for the in vitro motor activity of transcription termination factor Rho from Escherichia coli, Methods Mol Biol, vol.587, pp.137-154, 2010.
URL : https://hal.archives-ouvertes.fr/hal-00623571

F. D'heygere, A. Schwartz, and F. Coste, Monitoring RNA unwinding by the transcription termination factor rho from Mycobacterium tuberculosis, Methods Mol Biol, vol.1259, pp.293-311, 2015.

I. Artsimovitch and R. Landick, Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals, Proc Natl Acad Sci U S A, vol.97, pp.7090-7095, 2000.

W. Nowatzke, L. Richardson, and J. P. Richardson, Purification of transcription termination factor Rho from Escherichia coli and Micrococcus luteus, Methods Enzymol, vol.274, pp.353-363, 1996.

M. Kashlev, E. Nudler, and K. Severinov, Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase, Methods Enzymol, vol.274, pp.326-334, 1996.

M. Rabhi, V. Gocheva, and F. Jacquinot, Mutagenesis-based evidence for an asymmetric configuration of the ring-shaped transcription termination factor Rho, J Mol Biol, vol.405, pp.497-518, 2011.
URL : https://hal.archives-ouvertes.fr/hal-00608241

M. Rabhi, A. R. Rahmouni, and M. Boudvillain, Transcription termination factor Rho: a ring-shaped RNA helicase from bacteria, Jankowsky E (ed) RNA helicases, vol.19, pp.243-271, 2010.
URL : https://hal.archives-ouvertes.fr/hal-00610004

V. Gocheva, L. Gall, A. Boudvillain, and M. , Direct observation of the translocation mechanism of transcription termination factor Rho, Nucleic Acids Res, vol.43, pp.2367-2377, 2015.
URL : https://hal.archives-ouvertes.fr/hal-01171254

T. Soper, P. Mandin, and N. Majdalani, Positive regulation by small RNAs and the role of Hfq, Proc Natl Acad Sci U S A, vol.107, pp.9602-9607, 2010.

W. S. Yarnell and J. W. Roberts, Mechanism of intrinsic transcription termination and antitermination, Science, vol.284, pp.611-615, 1999.

C. Nadiras,

, Apport personnel : Article II ________________ ____________________________________ 170