Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements.

Abstract

The in vitro formation of transcription complexes with Escherichia coli RNA polymerase was monitored using fluorescence anisotropy measurements of labeled fragments of DNA. The multicomponent system consisted of holo or core RNA polymerase (RNAP) and lac or gal promoter fragments of DNA (in different configurations), in the presence or absence of CRP activator protein (wt or mutants) with its ligand, cAMP. Values of the apparent binding constants characterizing the system were obtained, as a result of all processes taking place in the system. The interaction of the promoters with core RNAP in the absence of CRP protein was characterized by apparent binding constants of 0.67 and 1.9 x 10(6) M(-1) for lac166 and gal178, respectively, and could be regarded as nonspecific. The presence of wt CRP enhanced the strength of the interaction of core RNAP with the promoter, and even in the case of gal promoter it made this interaction specific (apparent binding constant 2.93 x 10(7) M(-1)). Holo RNAP bound the promoters significantly more strongly than core RNAP did (apparent binding constants 1.46 and 40.14 x 10(6) M(-1) for lac166 and gal178, respectively), and the presence of CRP also enhanced the strength of these interactions. The mutation in activator region 1 of CRP did not cause any significant disturbances in the holo RNAP-lac promoter interaction, but mutation in activator region 2 of the activator protein substantially weakened the RNAP-gal promoter interaction.

Versatility of Cooperative Transcriptional Activation: A Thermodynamical Modeling Analysis for Greater-Than-Additive and Less-Than-Additive Effects

“…In a related study on several different E. coli promoters, RNAP concentrations were scaled up from about 0.1 to 1 M. The relevant dissociation constants were found to be in a similar range as the RNAP concentrations, namely, in the range of 0.01 to 10 M [47] . A more recent study based on fluorescence anisotropy measurements reports from a dissociation constant M for the lac promoter of E. coli and from RNAP concentrations that can be varied in a range of 0.1 to 1 M [48] . In our context, this would imply that varies from 0.1 to 1.…”

Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans

“…Crystallographic studies of E . coli CRP have been performed to determine the structure of a CRP-cAMP complex, its target DNA, and the carboxyl-terminal domain of the RNA polymerase (RNAP) α-subunit[ 15 – 19 ]. It is shown that CRP contains a helix-turn-helix DNA-binding motif in its carboxyl-terminal domain and a cAMP-binding site in its amino-terminal domain[ 20 – 22 ].…”

Fast and continuous-flow separation of DNA-complexes and topological DNA variants in microfluidic chip format

“…The built complexes are stable due to the high binding constants of YOYO-1, 6 Â 10 8 mol À1 , 41 and actinomycin D, 10 6 mol À1 , 42 the binding constant of E. coli RNA polymerase core enzyme is 1.16 Â 10 6 mol À1 . 43 Resolutions of 0.29 and 0.46 were achieved for complex separation. The run time for a gel was 3 h.…”