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Charles Yanofsky - One of the best experts on this subject based on the ideXlab platform.

  • analysis of tryptophanase operon expression in vitro accumulation of tnac peptidyl trna in a release Factor 2 depleted s 30 extract prevents Rho Factor action simulating induction
    Journal of Biological Chemistry, 2002
    Co-Authors: Feng Gong, Charles Yanofsky
    Abstract:

    Abstract Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. The key feature of this antitermination mechanism has been shown to be the retention of uncleaved TnaC-peptidyl-tRNA in the translating ribosome. This ribosome remains stalled at the tna stop codon and blocks the access of Rho Factor to the tnatranscript, thereby preventing transcription termination. In normal S-30 preparations, synthesis of a TnaC peptide containing arginine instead of tryptophan at position 12 (Arg12-TnaC) was shown to be insensitive to added tryptophan, i.e.Arg12-TnaC-peptidyl-tRNA was cleaved, and there was normal Rho-dependent transcription termination. When the S-30 extract used was depleted of release Factor 2, Arg12-TnaC-tRNAPro was accumulated in the absence or presence of added tryptophan. Under these conditions the accumulation of Arg12-TnaC-tRNAPro prevented Rho-dependent transcription termination, mimicking normal induction. Using a minimal in vitro transcription system consisting of a tna template, RNA polymerase, and Rho, it was shown that RNA sequences immediately adjacent to thetnaC stop codon, the presumed boxA andrut sites, contributed most significantly to Rho-dependent termination. The tna boxA-like sequence appeared to serve as a segment of the Rho “entry” site, despite its likeness to the boxA element.

  • the mechanism of tryptophan induction of tryptophanase operon expression tryptophan inhibits release Factor mediated cleavage of tnac peptidyl trnapro
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Feng Gong, Yoshikazu Nakamura, Charles Yanofsky
    Abstract:

    Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNAPro) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNAPro accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release Factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNAPro and release of TnaC from the ribosome-mRNA complex. Release Factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNAPro was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNAPro inhibits both TnaC peptidyl-tRNAPro hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho Factor binding and subsequent transcription termination.

  • Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.
    Journal of Biological Chemistry, 2000
    Co-Authors: Feng Gong, Charles Yanofsky
    Abstract:

    Abstract Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho Factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA′-′trpE fusion, we demonstrate that induction results in a 15–20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 oftnaC by an Arg codon, or changing the tnaCstart codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho Factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNAPro. Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducerin vitro. This tryptophan analog is not incorporated into TnaC.

  • evidence suggesting cis action by the tnac leader peptide in regulating transcription attenuation in the tryptophanase operon of escherichia coli
    Journal of Bacteriology, 1995
    Co-Authors: Kurt Gish, Charles Yanofsky
    Abstract:

    Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Elevated levels of tryptophan induce transcription antitermination at one or more Rho Factor-dependent termination sites in the leader region of the operon. Induction requires translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. In the present paper, we show that two bacterial species that lack tryptophanase activity, Enterobacter aerogenes and Salmonella typhimurium, allow tryptophanase induction and tna operon regulation when they carry a plasmid containing the E. coli tna operon. The role of tnaC in induction was examined by introducing mutations in a 24-nucleotide segment of tnaC of E. coli surrounding and including the crucial Trp codon 12. Some mutations resulted in a noninducible phenotype; these mostly introduced nonconservative amino acid substitutions in TnaC. Other mutations had little or no effect; these generally were in third positions of codons or introduced conservative amino acid replacements. A tryptophan-inserting, UGA-reading glutamine suppressor tRNA was observed to restore partial regulation when Trp codon 12 of tnaC was changed to UGA. Stop codons introduced downstream of Trp codon 12 in all three reading frames established that induction requires translation in the natural tnaC reading frame. Our findings suggest that the TnaC leader peptide acts in cis to prevent Rho-dependent termination.

  • Bicyclomycin sensitivity and resistance affect Rho Factor-mediated transcription termination in the tna operon of Escherichia coli.
    Journal of bacteriology, 1995
    Co-Authors: Charles Yanofsky, Virginia Horn
    Abstract:

    The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho Factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect Rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be required by mutations at any one of three loci, two of which appear to be Rho and rpoB.

Feng Gong - One of the best experts on this subject based on the ideXlab platform.

  • analysis of tryptophanase operon expression in vitro accumulation of tnac peptidyl trna in a release Factor 2 depleted s 30 extract prevents Rho Factor action simulating induction
    Journal of Biological Chemistry, 2002
    Co-Authors: Feng Gong, Charles Yanofsky
    Abstract:

    Abstract Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. The key feature of this antitermination mechanism has been shown to be the retention of uncleaved TnaC-peptidyl-tRNA in the translating ribosome. This ribosome remains stalled at the tna stop codon and blocks the access of Rho Factor to the tnatranscript, thereby preventing transcription termination. In normal S-30 preparations, synthesis of a TnaC peptide containing arginine instead of tryptophan at position 12 (Arg12-TnaC) was shown to be insensitive to added tryptophan, i.e.Arg12-TnaC-peptidyl-tRNA was cleaved, and there was normal Rho-dependent transcription termination. When the S-30 extract used was depleted of release Factor 2, Arg12-TnaC-tRNAPro was accumulated in the absence or presence of added tryptophan. Under these conditions the accumulation of Arg12-TnaC-tRNAPro prevented Rho-dependent transcription termination, mimicking normal induction. Using a minimal in vitro transcription system consisting of a tna template, RNA polymerase, and Rho, it was shown that RNA sequences immediately adjacent to thetnaC stop codon, the presumed boxA andrut sites, contributed most significantly to Rho-dependent termination. The tna boxA-like sequence appeared to serve as a segment of the Rho “entry” site, despite its likeness to the boxA element.

  • the mechanism of tryptophan induction of tryptophanase operon expression tryptophan inhibits release Factor mediated cleavage of tnac peptidyl trnapro
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Feng Gong, Yoshikazu Nakamura, Charles Yanofsky
    Abstract:

    Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNAPro) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNAPro accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release Factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNAPro and release of TnaC from the ribosome-mRNA complex. Release Factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNAPro was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNAPro inhibits both TnaC peptidyl-tRNAPro hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho Factor binding and subsequent transcription termination.

  • Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.
    Journal of Biological Chemistry, 2000
    Co-Authors: Feng Gong, Charles Yanofsky
    Abstract:

    Abstract Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho Factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA′-′trpE fusion, we demonstrate that induction results in a 15–20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 oftnaC by an Arg codon, or changing the tnaCstart codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho Factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNAPro. Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducerin vitro. This tryptophan analog is not incorporated into TnaC.

Marc Boudvillain - One of the best experts on this subject based on the ideXlab platform.

  • Monitoring RNA unwinding by the transcription termination Factor Rho from Mycobacterium tuberculosis.
    Methods in Molecular Biology, 2014
    Co-Authors: François D’heygère, Annie Schwartz, Franck Coste, Bertrand Castaing, Marc Boudvillain
    Abstract:

    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.

  • Terminator still moving forward: expanding roles for Rho Factor
    Current Opinion in Microbiology, 2013
    Co-Authors: Marc Boudvillain, Nara Figueroa-bossi, Lionello Bossi
    Abstract:

    Rho Factor is a molecular motor that translocates along nascent RNA and acts on the transcription elongation complex to promote termination. Besides contributing to transcriptional punctuation of the bacterial genome, Rho can act intragenically under conditions that perturb coupling of translation and transcription. Recent advances have shed new light onto several aspects of Rho function, including the translocation mechanism, the avoidance of potential conflicts between DNA replication and transcription, suppression of pervasive antisense transcription and recruitment in riboswitch and small RNA-dependent regulation. Altogether, these findings further highlight the relevance of Rho Factor, both as a multi-task housekeeper and gene regulator.

  • A stepwise 2 '-hydroxyl activation mechanism for the bacterial transcription termination Factor Rho helicase
    Nature Structural and Molecular Biology, 2009
    Co-Authors: Annie Schwartz, A. Rachid Rahmouni, Makhlouf Rabhi, Frederique Jacquinot, Emmanuel Margeat, Marc Boudvillain
    Abstract:

    The bacterial Rho Factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho-RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large(similar to 7-nucleotide-long) discrete steps triggered by 2'-hydroxyl activation events. This periodic 2'-OH-dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.

Paul Gollnick - One of the best experts on this subject based on the ideXlab platform.

  • trna trp translation of leader peptide codon 12 and other Factors that regulate expression of the tryptophanase operon
    Journal of Bacteriology, 1990
    Co-Authors: Paul Gollnick, Charles Yanofsky
    Abstract:

    Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan. This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon. Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction. We have used site-directed mutagenesis to investigate the role of the single Trp codon, at position 12 in tnaC, in regulation of the operon. Codon 12 was changed to either a UAG or UGA stop codon or to a CGG arginine codon. Induction by tryptophan was eliminated by any of these changes. Studies with suppressor tRNAs indicated that tRNA(Trp) translation of codon 12 in tnaC is essential for induction of the operon. Reduction of tna expression by a miaA mutation supports a role for translation by tRNA(Trp) in regulation of the operon. Frameshift mutations and suppression that allows translation of tnaC to proceed beyond the normal stop codon result in constitutive tna operon expression. Deletion of a potential site for Rho Factor utilization just beyond tnaC also results in partial constitutive expression. These studies suggest possible models for tryptophan induction of tna operon expression involving tRNA(Trp)-mediated frame shifting or readthrough at the tnaC stop codon.

John P. Richardson - One of the best experts on this subject based on the ideXlab platform.

  • How Rho exerts its muscle on RNA.
    Molecular Cell, 2006
    Co-Authors: John P. Richardson
    Abstract:

    Rho Factor in bacteria terminates transcription by using energy from ATP hydrolysis to forcefully dissociate the transcripts from RNA polymerase. Adelman et al. (2006) used data from presteady-state ATPase kinetics to support a rational mechanistic model for Rho's action on RNA.

  • Transcription Factor Rho does not require a free end to act as an RNA-DNA helicase on an RNA.
    Journal of Biological Chemistry, 2001
    Co-Authors: Brandt R. Burgess, John P. Richardson
    Abstract:

    Abstract Escherichia coli Rho Factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5′ terminus. To determine whether Rho's activity is 5′-end-dependent, its ability to bind to and function on a circular derivative of λ cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of λ cro RNA containing an extra 10-nucleotide sequence near the 5′-end that was complementary to a sequence located near the 3′-end. Rho bound nearly as tightly to the circular derivative RNA as to the standardcro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5′ terminus.

  • RNA passes through the hole of the protein hexamer in the complex with the Escherichia coli Rho Factor.
    Journal of Biological Chemistry, 2000
    Co-Authors: Brandt R. Burgess, John P. Richardson
    Abstract:

    Abstract Escherichia coli transcription termination Factor Rho is a ring-shaped hexameric protein that uses the energy derived from ATP hydrolysis to dissociate RNA transcripts from the ternary elongation complex. To test a current model for the interaction of Rho with RNA, three derivatives of Rho were made containing single cysteine residues and modified with a photo-activable cross-linker. The positions for the cysteines were: 1) in part of the primary RNA-binding site in the N terminus (Cys-82 Rho); 2) in a connecting polypeptide proposed to be on the outside of the hexamer (Cys-153 Rho); and 3) near the proposed secondary RNA-binding site in the ATP-binding domain (Cys-325 Rho). Results from the cross-linking of the modified Rho proteins to a series of λ cro RNA derivatives showed that Cys-82 Rho formed cross-links with all transcripts containing the Rho utilization (rut) site, that Cys-325 Rho formed cross-links to transcripts that had therut site and 10 or more residues 3′ of the rut site, and that Cys-153 did not form cross-links with any of the transcripts. From a model of the quaternary structure of Rho, which is largely based on homology to the F1-ATPase, amino acid 82 is located near the top of the hexamer, and amino acid 325 is located on a solvent-accessible loop in the center of the hexamer. These data are consistent with binding of the rut region of RNA around the crown, with its 3′-segment passing through the center of the Rho hexamer.

  • The NMR structure of the RNA binding domain of E. coli Rho Factor suggests possible RNA-protein interactions.
    Nature Structural Biology, 1998
    Co-Authors: Deborah M. Briercheck, John P. Richardson, Todd C. Wood, Timothy J. Allison, Gordon S. Rule
    Abstract:

    The NMR structure of the RNA binding domain of E.coli Rho Factor suggests possible RNA–protein interactions

  • Rho-dependent Termination of Transcription Is Governed Primarily by the Upstream Rho Utilization (rut) Sequences of a Terminator
    Journal of Biological Chemistry, 1996
    Co-Authors: Lislott V. Richardson, John P. Richardson
    Abstract:

    Abstract A Rho-dependent transcription terminator in Escherichia coli DNA consists of an upstream part for Rho utilization (rut) and the transcription stop point (tsp) region. To test the role of the tsp region variants of the coliphage λ cro gene terminator, tR1, containing inserts of non-terminator sequences between its rut and tsp regions were tested for termination function. The results showed that termination occurred with high efficiency at multiple sites in each of the new sequences with the positions of the sites coinciding with transcriptional pause points in the insert sequence and that the efficiency of termination was not directly proportional to the extent of pausing at those points. Thus, in contrast to the rut sequences, which are relatively rare in DNA, many different sequence segments can function as a tsp region. Studies with isolated transcripts showed that a rut element and sequences 3′ of the rut element were both needed to activate ATP hydrolysis by Rho Factor with the degree of activation depending on the length and the specific sequence of the 3′ segment. These results support models for Rho action in which ATP hydrolysis is coupled to interactions of Rho protein with RNA 3′ of the rut region.