Photoaffinity labeling of the tetracycline binding site of the Escherichia coli ribosome : The uses of a high intensity light source and of radioactive sancycline derivatives

Abstract

[ 3H]Tetracycline (TC) has been shown to photoincorporate into the Escherichia coli ribosome. However, the utility of this process for characterizing the TC binding site on the ribosome is diminished by competing side reactions which also lead to incorporation of radioactivity. In this work we first conducted a detailed study of the labeling processes occurring when ribosomes are irradiated in the presence of [ 3H]TC with a common, rather low intensity, lamp. On the basis of the results of this study we next explored the usefulness of photoaffinity labeling of the TC site of both irradiation with a high-intensity laser and radioactive, functional TC derivatives having different photochemical properties than TC itself. Labeling patterns determined by polyacrylamide gel electrophoretic analysis of ribosomal proteins extracted from photoaffinity-labeled 30S subunits provided strong evidence that these two approaches offer distinct advantages for characterizing the TC binding site