Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps

The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C. The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this three-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.     

Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime: application to kinetics of open complex formation between Esigma70 RNA polymerase and lambdaP(R) promoter DNA

A novel analytical method based on the exact solution of equations of kinetics of unbranched first- and pseudofirst-order mechanisms is developed for application to the process of Esigma70 RNA polymerase (R)-lambdaPR promoter (P) open complex formation, which is described by the minimal three-step mechanism with two kinetically significant intermediates (I1, I2), [equation: see text], where the final product is an open complex RPo. The kinetics of reversible and irreversible association (pseudofirst order, [R] > [P]) to form long-lived complexes (RPo and I2) and the kinetics of dissociation of long-lived complexes both exhibit single exponential behavior. In this situation, the analytical method provides explicit expressions relating observed rate constants to the microscopic rate constants of mechanism steps without use of rapid equilibrium or steady-state approximations, and thereby provides a basis for interpreting the composite rate constants of association (ka), isomerization (ki), and dissociation (kd) obtained from experiment for this or any other sequential mechanism of any number of steps. In subsequent papers, we apply this formalism to analyze kinetic data obtained in the reversible and irreversible binding regimes of Esigma70 RNA polymerase (R)-lambdaP(R) promoter (P) open complex formation.     

How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter

We previously described the structures and functions of specific complexes between the bla promoter from Tn3 (present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval-Valentin and Ehrlich, 1988). We report here that the 2:1 complex cannot be detected below 25 degrees C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30 degrees C, the amounts of both species become equal for RNAP/promoter ratio r30 less than or equal to r less than or equal to 70. The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay. Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre-incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex. We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25 degrees C. A possible mechanism of formation of the 2:1 complex is outlined. In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.     

Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunit

Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.     

Strand opening-deficient Escherichia coli RNA polymerase facilitates investigation of closed complexes with promoter DNA: effects of DNA sequence and temperature

Formation of the strand-separated, open complex between RNA polymerase and a promoter involves several intermediates, the first being the closed complex in which the DNA is fully base-paired. This normally short lived complex has been difficult to study. We have used a mutant Escherichia coli RNA polymerase, deficient in promoter DNA melting, and variants of the P(R) promoter of bacteriophage lambda to model the closed complex intermediate at physiologically relevant temperatures. Our results indicate that in the closed complex, RNA polymerase recognizes base pairs as double-stranded DNA even in the region that becomes single-stranded in the open complex. Additionally, a particular base pair in the -35 region engages in an important interaction with the RNA polymerase, and a DNase I-hypersensitive site, pronounced in the promoter DNA of the open complex, was not present. The effect of temperature on closed complex formation was found to be small over the temperature range from 15 to 37 degrees C. This suggests that low temperature complexes of wild type RNA polymerase and promoter DNA may adequately model the closed complex.     

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.     

Effect of temperature on the transcription by bacteriophage T3-induced RNA polymerase

Bacteriophage T3-induced RNA polymerase is rapidly inactivated at 42 degrees C. Addition of T3 DNA delays this process for 30 s and reduces the rate with which the enzyme activity is lost indicating that a labile binary complex between T3 DNA and polymerase must have been formed. The ternary complex between T3-specific RNA polymerase, T3 DNA, and nascent RNA chains obtained when the enzyme is incubated with T3 DNA, GTP, ATP, and UTP is stable to heat (42 degrees C) and only slowly inactivated by polyvinyl sulfate. The optimal temperature for the formation of polyanionresistant ternary complexes is 30 degrees C while the elongation of T3 RNA chains proceeds fastest at 38 degrees C.     

Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)