A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 16S rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA(UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L1I, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 16S rRNA (small subunit) mutant ofEscherichia coli that suppressed UGA mutations and implicated in particular the universally conserved nucleotide C1054, located in the conserved helix 34 (1). A key role for 23S rRNA (large ribosomal subunit) in peptide bond formation, translocation, and translational accuracy has been clearly established (refs. 2-5 and references therein). The small ribosomal subunit, on the other hand, has generally been thought to be the subunit involved in codon-specific translational events in both elongation and termination. Indeed, until recently, the majority of rRNA suppressors of nonsense (termination codon) mutations obtained were associated with 16S rRNA (6). In this paper, however, we describe a 23S rRNA nonsense suppressor that works at UGA mutant codons in trpA but not at UAA or UAG mutations at the same codon positions. A preliminary report of these results was presented at the Cold Spring Harbor Laboratory meeting on the Mo-
Point mutations were produced near the 3' end of E. coli 16S rRNA by bisulfite mutagenesis in a 121 base loop-out (1385 to 1505) in a heteroduplex of wild type (pKK3535) and deletion mutant plasmids. Two highly conserved, single stranded regions flank an irregular helix (1409-1491) in the area studied. Only a single mutation was isolated in the flanking regions, a transition at C1402, (normally methylated on the base and ribose in rRNA). Mutations occurred throughout the irregular helix. All mutant rRNAs were processed and assembled into 30S subunits capable of interacting with 50S subunits. Growth rates ranged from faster to significantly slower than cells with the wild type transcript. In particular, mutations at C1467 or C1469 cause slow growth. These two transitions (in a bulge region within the helix) reduced the bulge by additional base pairing.
Mutations were constructed at three sites in 16S rRNA in E. coli by oligonucleotide-directed mutagenesis, and cloned into the rrnB operon on either pKK3535 or pNO2680. The mutated bases, G966, C967, and G1207, are located in the 3' major domain of 16S rRNA and are sites post-transcriptionally modified by methylation. We constructed a deletion mutation at C967 (delta 967) and three substitution mutations at each of the following sites: G966, C967, and G1207. By maxicell analysis, we found that all of the mutations were processed normally and incorporated into 30S subunits and 70S ribosomes. We found that delta 967 was a dominant lethal mutation while the substitution mutations at G966 and C967 had no effects on cell growth rate. The mutants C1207 and U1207 were shown to have dominant lethal phenotypes while A1207 had no effect on cell growth rate. These results help to establish the importance of methyl-modified regions to ribosome function.
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