REGIONS OF 23-S RIBOSOMAL-RNA PROXIMAL TO TRANSFER-RNA BOUND AT THE P-SITES AND E-SITES

tRNA(Phe) transcribed in a T7 RNA polymerase system has been modified in such a way that 4-thiouridines have randomly replaced unmodified ur idines. These 4-thiouridines serve as sites for conjugation of the cle avage reagent 5-iodoacetamido-1,10-phenanthroline (IOP). 1,10-Phenanth oline, when complexed with Cu2+ in a reducing environment, causes hydr olysis of nearby nucleic acids. We show here that tRNA-phenanthroline (tRNA-OP) conjugates, when bound in situ to the P- and E-sites of 70 S ribosomes, cause cleavage, mainly in domains I, III and V of 23 S rib osomal RNA (rRNA). The cleavage sites in domain V predominantly occur very close to or in the peptidyl-transferase region. The regions of do main I and III that are cleaved are apparently folded in the 50 S ribo somal subunit so as to be proximal to the peptidyl-transferase center. Most of the cleavage events occur whether the tRNA-OP conjugate is bo und to ribosomes alone, or yeast tRNA is also present in the P/P hybri d state. Cleavages that occur only in the absence of yeast tRNA are li mited to the 1100 region of domain II, and the 2800 region of domain V I. Cleavages that occur only in the presence of yeast occur in the 217 0 region of domain V. The regions of 23 S rRNA in which tRNA-OP induce d cleavage occur complement those sites shown by chemical protection a nd cross-liking to be in a close proximity to the tRNA. However, the c leavage approach allows a more versatile and expanded view of the near neighborhood of rRNA surrounding the tRNA. These results provide cons iderable information which will allow a more detailed modeling of the tertiary structure of the 50 S ribosomal subunit. (C) 1995 Academic Pr ess Limited