Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 S ribosomal RNA.

RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macromolecules. C1054, a virtually universally conserved nucleotide in the 16 S (small subunit) ribosomal RNA of Escherichia coli, is part of a highly conserved bulge in helix 34, which has been located at the decoding site of the ribosome. This helix has been implicated in several translational events, including peptide chain termination and decoding accuracy. Here, we observed interesting differences in phenotype associated with the three base substitutions at, and the deletion of, nucleotide C1054. The phenotypes examined include suppression of nonsense codons on different media and at different temperatures, lethality conditioned by temperature and level of expression of the mutant rRNA, ribosome profiles upon centrifugation through sucrose density gradients, association of mutant 30 S subunits with 50 S subunits, and effects on the action of tRNA suppressor mutants. Some of our findings contradict previously reported properties of individual mutants. Particularly notable is our finding that the first reported 16 S rRNA suppressor of UGA mutations was not a C1054 deletion but rather the base substitution C1054A. After constructing deltaC1054 by site-directed mutagenesis, we observed, among other differences, that it does not suppress any of the trpA mutations previously reported to be suppressed by the original UGA suppressor. In general, our results are consistent with the suggestion that the termination codon readthrough effects of mutations at nucleotide 1054 are the result of defects in peptide chain termination rather than of decreases in general translational accuracy. The phenotypic heterogeneity associated with different mutations at this one nucleotide position may be related to the mechanisms of involvement of this nucleotide, the two-nucleotide bulge, and/or helix 34 in particular translational events. In particular, previous indications from other laboratories of conformational changes associated with this region are consistent with differential effects of 1054 mutations on RNA-RNA or RNA-protein interactions. Finally, the association of a variety of phenotypes with different changes at the same nucleotide may eventually shed light on speculations about the coevolution of parts of ribosomal RNA with other translational macromolecules.

Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA.

To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3' domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within the 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UGA, through suppression of UAG and UGA, but not UAA, to suppression of all three termination codons. The two helices in 16S rRNA have previously been associated both physically and functionally with the decoding center of the ribosome. The 23S region is part of the binding site for the large subunit protein L11 and the antibiotic thiostrepton, both of which have been shown to affect peptide chain termination. Finally, we have demonstrated that the 23S mutant A1093, which suppresses trpA UGA mutations very efficiently, is lethal at temperatures above 36 degrees C (when highly expressed). This lethality is overcome by secondary 23S rRNA mutations in domain V. Our results suggest that specific regions of 16S and 23S rRNA are involved in peptide chain termination, that the lethality of A1093 is caused by high-level UGA suppression, and that intramolecular interaction between domains II and V of 23S rRNA may play a role in peptide chain termination at the UGA stop codon.

Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination.

Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination. Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204). Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination. These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation.

A radioactive assay for the physiological activity of the tryptophan synthetase alpha subunit in crude extracts of Escherichia coli.

A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to Trp, of the enzyme tryptophan synthetase. The assay may be applied to crude bacterial extracts, and is based on the measurement of incorporation of radioactivity from [3H]Ser into Trp. Comparison with previous colorimetric assays indicates an improvement in sensitivity of about 30-fold, and advantages in terms of sample economy and simplified manipulation.

Third position base changes in codons 5' and 3' adjacent UGA codons affect UGA suppression in vivo.

The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency. However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon. These conclusions are drawn from experiments in which the two Ser codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coli have been replaced with other Ser codons by site-directed mutagenesis. Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT. We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA.

Reversion of trpA nonsense mutations by deletion of the chain-termination codons.

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.

Mutant 16S ribosomal RNA: a codon-specific translational suppressor.

We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211). The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4. It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations. The suppressor also does not allow mistranslation of the UGA-related trpA missense mutations UGG at positions 211 and 234, AGA at 211 and 234, CGA at 211, or UGU and UGC at 234. The suppressor mutation was mapped by genetic procedures to position 89 on the E. coli genetic map. Localization of the suppressor mutation to rrnB was achieved by cloning it in the low-copy-number plasmid pEJM007 by in vivo recombination from the chromosome. Recloning in bacteriophage M13 and subsequent DNA sequence analysis allowed the identification of the suppressor mutation as a deletion of the cytidylic acid residue at nucleotide position 1054 of the 16S ribosomal RNA. The mutant EcoRI-Xba I fragment from the suppressor gene was recloned, from M13, in an otherwise wild-type rrnB in the plasmid pEJM007, and UGA suppression was examined. The UGA-suppressing activity of the reconstructed suppressor-containing pEJM007 was indistinguishable from that of the original recombinant suppressor-containing plasmid. This result demonstrates that the C1054 deletion in 16S rRNA is both necessary and sufficient for UGA suppression. The existence of this mutant suggests an important role for rRNA in codon recognition, at least for accurate polypeptide chain termination.

Codon context effects in missense suppression.

After our first observation of codon context effects in missense suppression ( Murgola & Pagel , 1983), we measured the suppression of missense mutations at two positions in trpA in Escherichia coli. The suppressible codons in the trpA messenger RNA were the lysine codons, AAA and AAG, and the glutamic acid codons, GAA and GAG. The mRNA sites of the codons correspond to amino acids 211 and 234 of the trpA polypeptide, positions at which glycine is the wild-type amino acid. Our data demonstrated codon context effects with both pairs of codons. The results indicate that suppression of AAA and AAG by mutant lysine transfer RNAs was more efficient at 211 than at 234, whereas suppression of GAA and GAG by two different mutant glycine tRNAs was more efficient at 234 than at 211. In general, the context effects were more pronounced with GAG and AAG than with GAA and AAA. (In some instances it appeared that suppression of GAA or AAA at a given position was more effective than suppression of GAG or AAG.) By contrast, no context effects were observed with a glyT suppressor of AAA and AAG, a glyT GAA/G-suppressor, and a glyU suppressor of GAG. Our observation of this phenomenon in missense suppression demonstrates that codon context can affect polypeptide elongation and that the effects can be different depending on the codons and tRNAs examined. It is suggested that tRNA-tRNA interaction on the ribosome is involved in the observed context effects.

Missense and nonsense suppressors derived from a glycine tRNA by nucleotide insertion and deletion in vivo.

Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia coli and each containing an extra nucleotide in the anticodon loop, we generated new suppressors in vivo by spontaneous deletion of specific nucleotides from the anticodon loop. In one experiment, the new suppressor was generated by a double mutational event, base substitution and nucleotide deletion. A novel ochre suppressor is also described. It is very efficient in nonsense suppression but has no ms2i6 modification of the A residue on the 3' side of the anticodon. The results have important implications for tRNA structure-function relationships, tRNA recognition by tRNA-modifying enzymes, mechanisms of deletion mutation, and tRNA evolution.

Anticodon shift in tRNA: a novel mechanism in missense and nonsense suppression.

In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an extra adenylate residue in the anticodon loop of an Escherichia coli glycine tRNA was described. In this study, we provide in vivo evidence that the additional nucleotide causes an "anticodon shift" by one nucleotide in the 3' direction and that the "new" anticodon can explain the unanticipated coding properties of the suppressor. We converted the UGG suppressor with ethyl methanesulfonate, a base-substitution mutagen, to suppressors that read codons related to UGG by a single base change. Sequence analysis of each mutant tRNA revealed that its mutational alteration was an anticipated base change in one of the three nucleotides of the "new" anticodon. Although the new suppressors read codons beginning with A or U, the mutant tRNAs lack the customary hypermodified nucleosides on the 3' side of the anticodon. As determined on the basis of their in vivo coding specificities, the new mutant tRNAs do not continue to utilize the original anticodon triplet for decoding. Furthermore, the failure of the UGG suppressor to correct frameshift mutations throughout each of three genes of the trp operon suggests that the addition of a nucleotide to the anticodon loop of a tRNA does not necessarily result in out-of-frame decoding by the tRNA. Therefore, a "frameshift" mutation in a tRNA has principally changed the triplet codon recognition properties of the molecule.