CHEMICAL MODIFICATION INSITU OF ESCHERICHIA-COLI 30-S RIBOSOMAL-PROTEINS BY THE SITE-SPECIFIC REAGENT PYRIDOXAL-PHOSPHATE - INACTIVATION OF THE AMINOACYL-TRANSFER RNA AND MESSENGER-RNA BINDING-SITES

Abstract

.epsilon.-Amino groups of lysines of 30S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 > S1 > S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with A site than with P site binding. Initiation complex formation with some mRNA species (e.g., phage MS2 RNA) was affected more than with others (e.g., phage 17 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of inhibition; a role was also played by ribosomal proteins S1, S2 and S21. Substrate protection experiments showed that the 30S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.