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 R NA polymerase (RNAP) have been proposed to be essential in RNAP activa tion by CRP in type I promoters. These two proteins were shown to inte ract 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-s ubunit of RNAP (at position 321). The specific incorporation of fluore scence 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-T rp derivative of a protein even in the presence of high concentration of tryptophan containing protein(s). The CRP derivative is shown to re tain 100% of the native protein cAMP binding and specific DNA binding activity. Using a fluorescence polarization assay, it is also shown th at 5-OH-Trp derivative of CRP interacts with RNAP as well as the nativ e protein. The RNAP reconstituted with 5-OH-Trp derivative of the alph a-subunit retained the enzymatic activity. Fluorescence quenching stud ies show that Trp 321 of alpha-subunit is located in the region of the protein which is exposed to a solvent. These fluorescent derivatives of CRP and RNAP offer a great potential for studying the structural as pects of CRP-RNAP complex, the thermodynamics of complex formation, an d for monitoring the alterations of conformation of specific RNAP doma ins in response to CRP interaction at different stages of transcriptio n initiation.