4 NEWLY LOCATED PSEUDOURIDYLATE RESIDUES IN ESCHERICHIA-COLI 23S RIBOSOMAL-RNA ARE ALL AT THE PEPTIDYLTRANSFERASE CENTER - ANALYSIS BY THE APPLICATION OF A NEW SEQUENCING TECHNIQUE

A new technique has been developed for the facile location of pseudour idylate (PSI) residues in any RNA molecule. The method uses two known modification procedures which in combination uniquely identify U resid ues which have been converted into PSI. The first procedure involves r eaction of all U-like and G-like residues with yl-N'-beta-(4-methylmor pholinium)ethylcarbodiimide p-tosylate (CMC), followed by alkaline rem oval of all CMC groups except those linked to the N3 of PSI. This stop s reverse transcription, resulting in a gel band which identifies the U residue. The second procedure is uridine-specific hydrazinolysis whi ch cleaves the RNA chain at all U residues and produces a gel band upo n reverse transcription. PSI residues, being resistant to hydrazinolys is, are not cleaved and do not stop reverse transcription. This leads to the absence of a band at PSI residues. The combined method can also distinguish PSI from 5-methyluridine, 4-thiouridine, uridine-5-oxyace tic acid, and 2-thio-5-methylaminomethyluridine as shown by treating r RNA and tRNA species known to contain these modified bases at defined sites. By this procedure, four new sites for PSI in Escherichia coli 2 3S RNA were discovered, and one was disproven. The four new sites are at positions 2457, 2504, 2580, and 2605. The erroneous site is at posi tion 2555. These four new PSI residues, which are all in or within 2-3 residues of the peptidyltransferase ring, are thus in a position to p lay a functional and/or structural role at the peptidyltransferase cen ter. In addition, this work has shown the presence of a new unidentifi ed modified C residue at position 250 1, also in the peptidyltransfera se ring.