Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements.
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.
Bonarek
Piotr
P
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland. piotr.bonarek@uj.edu.pl
Kedracka-Krok
Sylwia
S
Kepys
Barbara
B
Wasylewski
Zygmunt
Z
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D005798:Genes, Bacterial; D007700:Kinetics; D007763:Lac Operon; D046911:Macromolecular Substances / Q000737:chemistry; D009154:Mutation; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D011953:Receptors, Cyclic AMP / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D011953:Receptors, Cyclic AMP; D011994:Recombinant Proteins; C047947:cyclic AMP receptor protein, E coli; D012321:DNA-Directed RNA Polymerases