Structural and functional implications in the eubacterial ribosome as revealed by protein-rRNA and antibiotic contact sites.
Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2-iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, and L36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at the peptide level in several proteins that were found to constitute the antibiotic-binding sites. Peptide 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-peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 with a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.
Wittmann-Liebold
B
B
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
Uhlein
M
M
Urlaub
H
H
Müller
E C
EC
Otto
A
A
Bischof
O
O
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001412:Bacillus subtilis / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000378:metabolism; D008969:Molecular Sequence Data; D010458:Peptidyl Transferases / Q000378:metabolism; D011691:Puromycin / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D015572:Spiramycin / Q000378:metabolism; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D011691:Puromycin; D015572:Spiramycin; D010458:Peptidyl Transferases