Energetic contributions to the initiation of transcription in E. coli.
The thermodynamics of RNA polymerase (RNAP) binding to a 108 base pair (bp) synthetic promoter with consensus sequences at the -35 and -10 bp binding regions upstream from the transcription start point were determined using isothermal titration calorimetry (ITC). The binding constant at 25 degrees C is 2.37+/-0.18x10(7) M(-1), which is reduced to 0.17+/-0.06x10(7) M(-1) with mutations in the -10 bp region but remained the same with mutations in the -35 binding region. The binding reactions were enthalpically-driven with exothermic binding enthalpies ranging from -57+/-6 kJ mol(-1) at 15 degrees C to -271+/-20 kJ mol(-1) at 35 degrees C yielding a large binding heat capacity change of -10.7+/-1.9 kJ mol(-1) K(-1), indicating a conformational change upon binding to the RNAP. Differential scanning calorimetry (DSC) scans of the thermal unfolding of RNAP and the promoter-RNAP complex exhibited an unfolding transition at 55.5+/-0.6 degrees C and at 58.9+/-0.5 degrees C for the RNAP but only one transition at 60.5+/-1.1 degrees C for the complex with van't Hoff enthalpy to transition enthalpy ratios of, resp., 3.2+/-0.3 and 4.3+/-0.5. The single transition of the complex results from a shift to 60.5 degrees C of the low temperature transition upon promoter binding to the structural unit unfolding at the lower temperature in RNAP. The large transition enthalpy ratios indicate that the sigma, alpha, alpha, beta, and beta' subunits unfold as almost independent entities. The dissociation thermodynamics of short transcription "bubble" duplexes of 7 promoters sequenced from -1 to -12 bp were determined from ITC and DSC measurements. The free energy change of the promoter binding to the RNAP and the free energy requirement for formation of the transcription bubble at the low promoter concentrations in the cell are sufficient to drive the initiation of transcription through the isomerization of the closed to the open form step of the RNAP-promoter complex.
Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Jayanthi
J
Ramprakash
Frederick P
FP
Schwarz
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017510:Protein Folding; D013816:Thermodynamics; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases