STRUCTURAL AND FUNCTIONAL IMPLICATIONS IN THE EUBACTERIAL RIBOSOME ASREVEALED BY PROTEIN-RIBOSOMAL-RNA AND ANTIBIOTIC CONTACT SITES

Contact sites between protein and rRNA in 30S and 50S ribosomal subuni ts of Escherichia coli and Bacillus stearothermophilus were investigat ed at the molecular level using UV and 2-iminothiolane as crosslinkers . Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, and L36) from these organisms were cross-linked in dire ct contact with the RNAs, and the peptide stretches as well as amino a cids involved were identified. Further, the binding sites of puromycin and spiramycin were established at the peptide level in several prote ins that were found to constitute the antibiotic-binding sites. Peptid e stretches of puromycin binding were identified from proteins S7, S14 , S18, L18, and L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA-pepti de contact sites with the peptides identified for antibiotic binding a nd with those altered in antibiotic-resistant mutants clearly showed i dentical peptide areas to be involved and, hence, demonstrated the fun ctional importance of these peptides. Further evidence for a functiona l implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacteriu m marismortui was incorporated into the E. coli ribosomes that were ac tive. The incorporated protein was present in 50S subunits and 70S par ticles, in disomes, and in higher polysomes. These results clearly dem onstrate the functional implication of protein L2 in protein biosynthe sis. Incorporation studies with a mutant of HmaL2 with a replacement o f histidine-229 by glycine completely abolished the functional activit y of the ribosome. Accordingly, protein L2 with histidine-229 is a cru cial element of the translational machinery.