Physical studies on interaction of transcription activator and RNA-polymerase: fluorescent derivatives of CRP and RNA polymerase.
Protein-protein interactions between cAMP receptor protein (CRP) and RNA polymerase (RNAP) have been proposed to be essential in RNAP activation by CRP in type I promoters. These two proteins were shown to interact in solution in the absence of promoter DNA (Heyduk et al., 1993). In this report we describe the preparation of fluorescent derivatives of CRP (fluorescent probes at position 13 and 85); and of the alpha-subunit of RNAP (at position 321). The specific incorporation of fluorescence probes was achieved by expressing protein in a bacteria strain, auxotrophic for tryptophan, in media containing 5-hydroxytryptophan (5-OH-Trp). The absorbance spectrum of a protein containing 5-OH-Trp is shifted towards longer wavelengths as compared to the native protein. This allows selective monitoring of the fluorescence signal of 5-OH-Trp derivative of a protein even in the presence of high concentration of tryptophan containing protein(s). The CRP derivative is shown to retain 100% of the native protein cAMP binding and specific DNA binding activity. Using a fluorescence polarization assay, it is also shown that 5-OH-Trp derivative of CRP interacts with RNAP as well as the native protein. The RNAP reconstituted with 5-OH-Trp derivative of the alpha-subunit retained the enzymatic activity. Fluorescence quenching studies show that Trp 321 of alpha-subunit is located in the region of the protein which is exposed to a solvent.(ABSTRACT TRUNCATED AT 250 WORDS)
Heyduk
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St. Louis University, Medical School, Department of Biochemistry and Molecular Biology, MO 63104.
Heyduk
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T
D006916:5-Hydroxytryptophan / Q000737:chemistry*; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D002373:Cyclic AMP Receptor Protein; D006916:5-Hydroxytryptophan; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases