Emanuel J. Murgola

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.

Evidence for a unique first position codon-anticodon mismatch in vivo☆

The Ser68(AGC) codon of the beta-lactamase gene was changed to the glycine codons GGA and GGC. With glycine at position 68, beta-lactamase is inactive because it does not have a nucleophilic side-chain to function in the reaction mechanism. The mutant SG68(GGA) allele had no detectable beta-lactamase activity; however, the mutant SG68(GGC) did produce a small amount of activity. Both mutant alleles produce comparable amounts of beta-lactamase protein in a maxi-cell system. To identify why these two same-sense beta-lactamase mutants differ phenotypically, we introduced the alleles into Escherichia coli strains with mutations that affect translational fidelity. The rpsD mutation, which decreases fidelity, significantly increased activity with the SG68(GGC) allele, while the rpsL mutation, which increases translational fidelity, had little effect on the beta-lactamase activity. The rpsD and rpsL alleles had no effect on the SG68(GGA) allele. From the allele specificity of the activity produced by the bla mutants, and from the differential effect of translational fidelity on the activity of the SG68(GGC) allele, we infer that tRNA(GCU)Ser, the AGU/C reading tRNA(Ser), mistranslates SG68(GGC) at a frequency of about 0.1%, and subsequently produces active beta-lactamase. This is the first observation of an A/G wobble with a wild-type tRNA at the first position of the codon-anticodon interaction.

Nucleotide substitution in the amino acid acceptor stem of lysine transfer RNA causes missense suppression

Previous results from this laboratory indicated that, in Escherichia coli K12, a new class of missense suppressors, which read the lysine codons AAA and AAG, may be misacylated lysine transfer RNAs. We therefore isolated and determined the nucleotide sequence of the lysine tRNA from two of the suppressor strains. In each case, we found both wild-type and mutant species of lysine tRNA, a result consistent with evidence that there are two genes for lysine tRNA in the E coli genome. The wild-type sequence was essentially identical to that reported for lysine tRNA from E. coli B. The mutant species isolated from each suppressor strain had a U for C70 nucleotide substitution, demonstrating that the AAG suppressor is a mutant lysine tRNA. The nucleotide substitution in the amino acid acceptor stem is consistent with the in vivo evidence that the suppressor corrects AAA and AAG missense mutations by inserting an amino acid other than lysine during polypeptide synthesis. This report represents the first verification of missense suppression caused by misacylation of a mutant tRNA.

Selection for new codons corresponding to position 234 of the tryptophan synthetase alpha chain of Escherichia coli

SummaryPrevious studies have demonstrated many amino acid and codon substitutions at position 211 of the alpha chain of tryptophan synthetase of Escherichia coli. In order to extend our studies on suppressor tRNAs and, in general, on accuracy in the translation of genetic information, we have devised specific selections for sense and nonsense codons corresponding to alpha chain position 234. Using codon-specific suppressors and selection for missense revertants of nonsense mutations, we have brought to 18 (from six) the number of codons at 234. This number includes all three nonsense codons and ten (of the remaining 15) that code for nonfunctional amino acids at that position. Furthermore, by adding to these results the testing of the suppressibility of trpA(234) nonsense mutations by suppressor tRNAs of known aminoacylation specificity, we have increased to 12 (from five) the number of amino acids whose functionality at position 234 is known.The existence of trpA mutants with so many amino acid substitutions at two positions in the tryptophan synthetase alpha chain should prove helpful for structure-function studies of that protein. Furthermore, the availability, at both positions, of at least 12 condons (including the three nonsense codons) that result in a nonfunctional alpha chain allows the selection of mutations that affect the suppression of a given codon as it occurs in two different mRNA contexts. Studies with such mutants should help elucidate the role in translational accuracy of mRNA secondary structure, tRNA-tRNA interactions on the ribosome, and interactions of the ribosome with tRNA, mRNA, and, in the case of nonsense suppression, peptidyl release factors.

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

SummaryBeginning 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.

Sequence analysis of the glyW region in Escherichia coli

We have determined the DNA sequence of a 1-kilobase segment of the Escherichia coli chromosome. The segment contains glyW, a duplicate gene for tRNA3Gly, and its flanking regions. An insertion sequence, previously known to have occurred spontaneously within the sequenced fragment, was identified as IS1. Possible sites for initiation and termination of transcription were identified by comparing them with the sequences of model promoter regions and termination structures. The results suggest that the expression of glyW may depend upon the expression of the preceding gene, pgsA, by transcriptional or translational overlap, by cotranscription of these two genes, or both.