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Nobuo Shimamoto - One of the best experts on this subject based on the ideXlab platform.

  • generality of the Branched Pathway in transcription initiation byescherichia coli rna polymerase
    Journal of Biological Chemistry, 2002
    Co-Authors: Motoki Susa, Ranjan Sen, Nobuo Shimamoto
    Abstract:

    Transcription initiation has been assumed to be a multi-step sequential process, although additional steps could exist. Initiation from the T7A1 promoter, in particular, apparently behavesin vitro in a manner that can be fully explained by the sequential Pathway. However, initiation from the λPRAL promoter has been shown to follow a Branched Pathway from which a part of the enzyme-promoter complex is arrested at the promoter raising the question as to which mechanism is general. We found that a moribund complex, characteristic of the arrested branch, is formed at the T7A1 promoter, especially in low salt condition indicating that the initiation mechanism for this promoter is also Branched. The results of DNA footprinting suggested that holoenzyme in the moribund complex is dislocated on DNA from the position of productive complex. However, only a small fraction of the binary complex becomes arrested at this promoter, and the interconversion between subspecies of binary complex is apparently more reversible than at the λPRAL promoter, which explains why the reaction Pathway appears to be sequential. These findings suggest a generality of the Branched Pathway mechanism, which would resolve contradictory observations that have been reported for various promoters.

  • a Branched Pathway in the early stage of transcription by escherichia coli rna polymerase
    Journal of Molecular Biology, 1996
    Co-Authors: Tomoko Kubori, Nobuo Shimamoto
    Abstract:

    The time-course of synthesis of long and short (abortive) transcripts by Escherichia coli RNA polymerase was investigated under single round conditions in vitro. The synthesis of long RNA initiated at the lambda PR (with an altered leader sequence) or at the lacUV5 promoter was completed within five minutes, but abortive transcripts were continuously synthesized for at least 20 minutes. The results indicate the presence of transcription complexes that are capable only of abortive synthesis, and not of productive elongation. Consistently, only one in four of the RNA polymerase molecules that initially associated with the lambda PR promoter synthesized long RNA. The enzyme reisolated from productive complexes synthesized both long and abortive transcripts, behaving just like the original enzyme. This suggests that RNA polymerase is homogeneous with respect to its ability to synthesize the two types of product. Overall, the results indicate that many transcription complexes can be irreversibly trapped in vitro in an abortive cycle.

Kasper Kirschner - One of the best experts on this subject based on the ideXlab platform.

  • the catalytic mechanism of tryptophan synthase from escherichia coli kinetics of the reaction of indole with the enzyme l serine complexes
    FEBS Journal, 2005
    Co-Authors: Andrew N Lane, Kasper Kirschner
    Abstract:

    The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme—L-serine complex, followed by C—C bond formation, reprotonation of the α carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme—L-serine complexes, leading to a Branched Pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4′ atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the α subunit on the reaction catalyzed by the β2 subunit.

  • the catalytic mechanism of tryptophan synthase from escherichia coli kinetics of the reaction of indole with the enzyme l serine complexes
    FEBS Journal, 2005
    Co-Authors: Andrew N Lane, Kasper Kirschner
    Abstract:

    The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a Branched Pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.

Andrew N Lane - One of the best experts on this subject based on the ideXlab platform.

  • the catalytic mechanism of tryptophan synthase from escherichia coli kinetics of the reaction of indole with the enzyme l serine complexes
    FEBS Journal, 2005
    Co-Authors: Andrew N Lane, Kasper Kirschner
    Abstract:

    The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme—L-serine complex, followed by C—C bond formation, reprotonation of the α carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme—L-serine complexes, leading to a Branched Pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4′ atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the α subunit on the reaction catalyzed by the β2 subunit.

  • the catalytic mechanism of tryptophan synthase from escherichia coli kinetics of the reaction of indole with the enzyme l serine complexes
    FEBS Journal, 2005
    Co-Authors: Andrew N Lane, Kasper Kirschner
    Abstract:

    The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a Branched Pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.

Stefano Bruno - One of the best experts on this subject based on the ideXlab platform.

  • Following ligand migration Pathways from picoseconds to milliseconds in type II truncated hemoglobin from Thermobifida fusca.
    PLoS ONE, 2012
    Co-Authors: Agnese Marcelli, Darío A. Estrin, Stefania Abbruzzetti, Alessandra Bonamore, Alberto Boffi, Alessandro Feis, Juan Pablo Bustamante, Cristina Gellini, Pier Remigio Salvi, Stefano Bruno
    Abstract:

    CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ∼100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ∼2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ∼100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ∼3 to ∼100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a Branched Pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb.

Agnese Marcelli - One of the best experts on this subject based on the ideXlab platform.

  • Following Ligand Migration Pathways from Picoseconds to Milliseconds in Type II Truncated Hemoglobin from
    2015
    Co-Authors: Thermobifida Fusca, Stefania Abbruzzetti, Alberto Boffi, Juan Pablo Bustamante, Agnese Marcelli, Ro Feis, Ra Bonamore, Cristina Gellini, Pier Remigio Salvi, Darío A. Estrin
    Abstract:

    CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to,100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of,2 ns representing almost 60 % of the overall reaction. An additional, small amplitude geminate recombination was identified at,100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from,3 to,100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a Branched Pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable o

  • Following ligand migration Pathways from picoseconds to milliseconds in type II truncated hemoglobin from Thermobifida fusca.
    PLoS ONE, 2012
    Co-Authors: Agnese Marcelli, Darío A. Estrin, Stefania Abbruzzetti, Alessandra Bonamore, Alberto Boffi, Alessandro Feis, Juan Pablo Bustamante, Cristina Gellini, Pier Remigio Salvi, Stefano Bruno
    Abstract:

    CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ∼100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ∼2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ∼100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ∼3 to ∼100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a Branched Pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb.