Fluorescence probes for studying the mechanisms of transcription activation

Tomasz Heyduk; Sandhya Callaci

doi:10.1117/12.182781

17 August 1994 Fluorescence probes for studying the mechanisms of transcription activation

Tomasz Heyduk, Sandhya Callaci

Proceedings Volume 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV; (1994) https://doi.org/10.1117/12.182781
Event: OE/LASE '94, 1994, Los Angeles, CA, United States

Abstract

Regulation of transcription involves a complex interplay between protein-ligand, protein-DNA, and protein-protein interactions. Fluorescence probes seem to be very well suited to study such complex systems since the selectivity and sensitivity of fluorescence makes possible to select only a part of the system for observation leaving the rest of it transparent to the technique. We have used fluorescence spectroscopy to study the activation of E.coli RNA polymerase by cAMP receptor protein (CRP). The cAMP interactions with CRP, domain flexibility in CRP molecule, the structure of CRP-DNA complex, and interaction of CRP with RNA-polymerase have been studied. Here we report the preparation and properties of 5-OH-Trp derivative of the sigma subunit of E.coli RNA polymerase. This subunit is responsible for specific promoter recognition. The obtained results show that the biological activities of the derivative are identical as observed for the native protein. Comparison of fluorescence properties of the 5-OH-Trp sigma derivative free and bound to the core RNA polymerase suggests a conformational change in the sigma protein induced by this interaction. These data show that replacement of Trp residues with 5-OH-Trp can be a very useful approach to prepare specific fluorescence derivatives of multimeric proteins.

Citation Download Citation

Tomasz Heyduk and Sandhya Callaci "Fluorescence probes for studying the mechanisms of transcription activation", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); https://doi.org/10.1117/12.182781

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