Base-pairing between 23S rRNA and tRNA in the ribosomal A site

The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-p-puromycin (s(4)TCPm) pho tochemically cross-links with high efficiency and specificity to G2553 of 2 3S rRNA and is peptidyl transferase reactive in its cross-linked state, est ablishing proximity between the highly conserved 2555 loop in domain V of 2 3S rRNA and the universally conserved CCA end of tRNA. To test for base-pai ring interactions between 23S rRNA and aminoacyl tRNA, site-directed mutati ons were made at the universally conserved nucleotides U2552 and G2553 of 2 3S rRNA in both E, coli and B. stearothermophilus ribosomal RNA and incorpo rated into ribosomes. Mutations at G2553 resulted in dominant growth defect s in E. coli and in decreased levels of peptidyl transferase activity in vi tro. Genetic analysis in vitro of U2552 and G2553 mutant ribosomes and CCA end mutant tRNA substrates identified a base-pairing interaction between C7 5 of aminoacyl tRNA and G2553 of 23S rRNA.