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We have isolated mutations in the Escherichia coli glnS gene encoding glutaminyl-tRNA synthetase [GlnS; L-glutamine:tRNAGln ligase (AMP-forming), EC 6.1.1.18] that give rise to gene products with altered specificity for tRNA and are designated "mischarging" enzymes. These were produced by nitrosoguanine mutagenesis of the glnS gene carried on a transducing phage (lambda pglnS+). We then selected for mischarging of su+3 tRNATyr with glutamine by requiring suppression of a glutamine-requiring beta-galactosidase amber mutation (lacZ1000). Three independently isolated mutants (glnS7, glnS8, and glnS9) were characterized by genetic and biochemical means. The enzymes encoded by glnS7, glnS8, and glnS9 appear to be highly selective for su+3 tRNATyr, because in vivo mischarging of other amber suppressor tRNAs was not detected. The GlnS mutants described here retain their capacity to correctly aminoacylate tRNAGln. All three independently isolated mutant genes encode proteins with isoelectric points that differ from those of the wild-type enzyme but are identical to each other. This suggests that only a single site in the enzyme structure is altered to give the observed mischarging properties. In vitro aminoacylation reactions with purified GlnS7 protein show that this enzyme can also mischarge some tRNA species lacking the amber anticodon. This is an example of mischarging phenotype conferred by a mutation in an aminoacyl-tRNA synthetase gene; the results are discussed in the context of earlier genetic studies with mutant tRNAs.

We infected various nonflagellated mutants of Escherichia coli with fla-transducing phages and followed the kinetics of the appearance of motility. Our analysis revealed two distinct classes of region III fla genes. Class II fla genes (hag, flaD) functioned 15 min later than class I fla genes (flaN, flaB, flaC, flaO, flaA, flbD, flaQ, flaP) in flagellar morphogenesis. We suggest that the two classes of fla genes are involved in two different stages, initiation (class I) and completion (class II), of flagellar formation.

The chloroplast gene rps12 for ribosomal protein S12 in a liverwort, Marchantia polymorpha, is split into three exons by two introns, one of which (intron 1) is discontinuous. Exon 1 of rps12 for the N-terminal portion of the S12 protein is far from exons 2 and 3 for the C-terminal portion on the opposite DNA strand. S1-nuclease protection analysis and Northern hybridization with RNA isolated from the liverwort chloroplasts showed that: (i) the exons 1 and 2-3 of the rps12 gene with the neighboring genes were transcribed separately, (ii) the trans-splicing of intron 1 occurred after the processing of two primary transcripts to two pre-mRNAs, and (iii) there was no particular order for the splicing of intron 1 (trans) and intron 2 (cis) in the rps12 gene. We propose a bimolecular interaction model for trans-splicing by assuming that intermolecular base pairings between two pre-mRNAs result in the formation of the structure typical of group II introns except for disruption in the loop III region. This structure could be constructed in intron 1 of tobacco rps12 gene.

We have isolated a lambda-transducing phage carrying the gene (glnS) for Escherichia coli glutaminyl-tRNA synthetase. The location of the glnS gene within the 13.5-kilobase E. coli DNA transducing fragment was determined by genetic means. The glnS gene was recloned into plasmid pBR322 and its nucleotide sequence was established. The DNA sequence translates to a protein of 550 amino acids.