Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.

The fidelity of protein biosynthesis in any cell rests on the accuracy of aminoacylation of tRNA. The exquisite specificity of this reaction is critically dependent on the correct recognition of tRNA by aminoacyl-tRNA synthetases. It is shown here that the relative concentrations of a tRNA and its cognate aminoacyl-tRNA synthetase are normally well balanced and crucial for maintenance of accurate aminoacylation. When Escherichia coli Gln-tRNA synthetase is overproduced in vivo, it incorrectly acylates the supF amber suppressor tRNA(Tyr) with Gln. This effect is abolished when the intracellular concentration of the cognate tRNA(Gln2) is also elevate. These data indicate that the presence of aminoacyl-tRNA synthetase and the cognate tRNAs in complexed form, which requires the proper balance of the two macromolecules, is critical in maintaining the fidelity of protein biosynthesis. Thus, limits exist on the relative levels of tRNAs and aminoacyl-tRNA synthetases within a cell.

Signal recognition particle (SRP) consists of a 7SL RNA molecule and 6 protein subunits. We have isolated and characterized cDNA clones from human liver which encode the 19kDa protein subunit (SRP19). This subunit binds to the RNA directly and mediates binding of a second polypeptide, the 54kDa subunit which is involved in signal sequence recognition. Amino acid sequences deduced from the human cDNA sequence were identical to amino acid sequences of tryptic peptides from canine pancreatic SRP19. In vitro transcription and translation of the human cDNA resulted in a protein product the same size as canine SRP19 which could be immunoprecipitated by an antiserum raised against canine SRP19. SRP19 synthesized in a cell-free system specifically bound to 7SL RNA. The sequence of SRP19 is discussed with respect to its binding to 7SL RNA.

We have isolated mutations in the Escherichia coli ginS gene encoding glutaminyl-tRNA synthetase [GinS; L-glutamine:tRNAGOn 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 ginS gene carried on a transducing phage (ApglnS+). We then selected for mischarging of su+3 tRNATYr with glutamine by requiring suppression of a glutamine-requiring .3-galactosidase amber mutation (lacZ,100). Three independently isolated mutants (glnS7, ginS8, and ginS9) were characterized by genetic and biochemical means. The enzymes encoded by ginS7, ginS8, and ginS9 appear to be highly selective for su+3 tRNAT , because in vivo mischarging of other amber suppressor tRNAs was not detected. The GlnS mutants described here retain their capacity to correctly aminoacylate tRNAGin. 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.

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