Diffusion-enhanced energy transfer shows accessibility of ribonucleic acid polymerase inhibitor binding sites

Rifamycin and Cibacron Blue F3GA are powerful inhibitors of Escherichia coli deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase. In addition, both inhibitors strongly absorb visible light, making them suitable for use as acceptors in energy-transfer experiments. Transfer of energy to these acceptors from freely diffusing energy donors with long excited-state lifetimes (approximately 10(-3) s) depends strongly on whether donor and acceptor can make direct intermolecular contact. We observe that the rate constant for energy transfer from a small terbium chelate to enzyme-bound rifamycin is 1 X 10(7) M-1 s-1, which is about half as large as the rate constant observed for free rifamycin in solution. This relatively small change upon binding indicates that enzyme-bound rifamycin is highly accessible to small molecules in the solvent. In the case of Cibacron Blue, under conditions where approximately 90% of this inhibitor is bound to RNA polymerase, the small amount of unbound inhibitor accounts for practically all of the observed energy transfer. This implies that enzyme-bound Cibacron Blue is relatively inaccessible to energy donors in the solution. The dependence of energy transfer on the accessibility of the acceptor is illustrated by using simple geometric models. synthesis of a stable, electrically neutral terbium chelate which can be efficiently excited with UV radiation is also described.