Abstract
Crosslinking of the 5'-anticodon base of ribosomal P site bound AcVal-tRNA to residue C-1400 of 16S RNA or to its equivalent in 18S RNA has been shown to occur on 70S or 80S ribosomes of both prokaryotes and eukaryotes [Ciesiolka, J., Nurse, K., Klein, J. and Ofengand, J. (1985) Biochemistry 24, 3233-3239]. In the present work, we show that the crosslinking rate, crosslinking yield, and site of crosslinking are all unchanged when the 50S subunit is omitted. Therefore, all of the positional features of tRNA-ribosome complexes which allow crosslinking to occur are entirely contained in the 30S subunit. Blockage of reverse transcription by crosslink formation was used to determine the site of crosslinking. This analysis revealed that RNA modifications which do not directly block base-pairing ligands sometimes allow the modified base to be transcribed, leading to doublet band formation even when there is only a single crosslink site.