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  • Title: Stopped-flow kinetic analysis of the interaction of Escherichia coli RNA polymerase with the bacteriophage T7 A1 promoter.
    Author: Johnson RS, Chester RE.
    Journal: J Mol Biol; 1998 Oct 23; 283(2):353-70. PubMed ID: 9769210.
    Abstract:
    We have conducted a detailed kinetic and thermodynamic analysis of open complex formation between Escherichia coli RNA polymerase and the A1 promoter from bacteriophage T7 by monitoring alterations in the intrinsic protein fluorescence of RNA polymerase in stopped-flow kinetic studies. The stopped-flow kinetic data are consistent with a minimal model involving four steps for the formation of the open complex. Arrhenius plots for both the association and dissociation reactions for the equilibrium binding step leading to the formation of the closed complex were linear. With a positive van't Hoff enthalpy (DeltaHobs=18(+/-3) kcal mol-1) and a positive entropy (DeltaSobs=94(+/-15) e.u.) change for the equilibrium binding process, formation of the closed complex is entropy driven. The value of the apparent association rate constant for this binding step was approximately three orders of magnitude less than that expected for facilitated binding. Thus, a minimum of two steps is required to describe the formation of the closed complex. A fast facilitated binding step appears to be followed by a conformational change in RNA polymerase which leads to the formation of the closed complex. A non-linear Arrhenius plot obtained for the isomerization step in the conversion of the closed complex to an open one indicates that there are at least two steps in the conversion of the closed complex to an open one. We have assigned the apparent activation energy of 9.1(+/-1.9) kcal mol-1 to the step involving a conformational change in the protein and nucleation of strand separation and the apparent activation energy of 46(+/-12) kcal mol-1 to the step involving strand separation. At 37 degreesC, the value of the macroscopic isomerization rate constant (0.26(+/-0.02) s-1) in the conversion of the closed complex to an open one was an order of magnitude greater than the value reported in abortive initiation assays. This suggests that open complex formation is not the rate-determining step in the initiation of transcription in the case of the A1 promoter. To gain greater insight into the mechanism of initiation at the A1 promoter, we investigated the process of abortive product formation (pppApU) under conditions of non-saturating concentrations of the initiating nucleotide. A comparison of the lag times in the approach to the steady-state rate of abortive product formation when the reaction was initiated by the addition of UTP, ATP, the enzyme and the A1 promoter, respectively, indicates that the initiating nucleotide plays a key regulatory role in the initiation of transcription in the case of the A1 promoter.
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