title: Kinetic studies and structural models of the association of E. coli sigma(70) RNA polymerase with the lambdaP(R) promoter: large scale conformational changes in forming the kinetically significant intermediates.
abstract: The kinetics of interaction of Esigma(70) RNA polymerase (R) with the lambdaP(R) promoter (P) were investigated by filter binding over a broad range of temperatures (7.3-42 degrees C) and concentrations of RNA polymerase (1-123 nM) in large excess over promoter DNA. Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I(2), RP(o)) are single-exponential with first order rate constant beta(CR). Interpretation of the polymerase concentration dependence of beta(CR) in terms of the three step mechanism of open complex formation yields the equilibrium constant K(1) for formation of the first kinetically significant intermediate (I(1)) and the forward rate constant (k(2)) for the conformational change converting I(1) to the second kinetically significant intermediate I(2): R + P-->(K(1))<--I(1)(k(2))-->I(2). Use of rapid quench mixing allows K(1) and k(2) to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing. Given the large (>60 bp) interface formed in I(1), its relatively small binding constant K(1) at 37 degrees C at this [salt] (approximately 6 x 10(6) M(-1)) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes. Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of k(a)[DeltaC(o,++)(a)= -1.5(+/-0.2)kcal K(-1)], now shown to originate directly from formation of I(1) [DeltaC(o)(1)= -1.4(+/-0.3)kcal K(-1)] rather than from the formation of I(2) as previously proposed. The isomerization I(1)-->I(2) exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy [E(act)(2)= 34(+/-2)kcal]. This kinetic signature suggests that formation of the transition state (I(1)-I(2)++ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I(1) involves coupled folding of unstructured regions of polymerase (beta, beta' and sigma(70)) and bending of promoter DNA (in the -10 region). We propose that interactions with region 2 of sigma(70) and possibly domain 1 of beta induce a kink at the -11/-12 base pairs of the lambdaP(R) promoter which places the downstream DNA (-5 to +20) in the jaws of the beta and beta' subunits of polymerase in I(1). These early interactions of beta and beta' with the DNA downstream of position -5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Saecker
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Tsodikov
authors.forename: Kristi L
authors.initials: KL
authors.lastname: McQuade
authors.forename: Peter E
authors.initials: PE
authors.lastname: Schlax
authors.forename: Michael W
authors.initials: MW
authors.lastname: Capp
authors.forename: M Thomas
authors.initials: MT
authors.lastname: Record
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics*; D003101:Collodion / Q000378:metabolism; D018983:DNA Footprinting; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014158:Transcription, Genetic
chemical_list: D004279:DNA, Viral; D012808:Sigma Factor; D003101:Collodion; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases