Peptide Mapping of Aminoacyl-tRNA Synthetases: Evidence for Internal Sequence Homology in Escherichia coli Leucyl-tRNA Synthetase

Abstract

Most aminoacyl-tRNA synthetases contain polypeptide chains of about either 50,000 or 100,000 daltons. Peptide mapping of tryptic, chymotryptic, or Staphylococcus aureus acid protease digests of seryl-tRNA synthetase (100,000, dimer) and leucyl-tRNA synthetase (100,000, monomer) from E. coli was done after selective modification of lysine residues with [ 14 C]succinic anhydride or of methionine residues with [ 14 C]iodoacetate. By use of thin-layer electrophoresis and chromatography on silicagel or cellulose plates followed by radioautography it was possible, depending upon the specific activity of the reagent used, to detect radioactive peptides obtained from as little as l μg of protein. Seryl-tRNA synthetase gave the correct number of tryptic peptides expected for a dimer of identical subunits. Leucyl-tRNA synthetase, on the other hand, gave roughly half the number of radioactive tryptic, chymotryptic, and acid protease peptides expected from the lysine, arginine, and methionine content of the 100,000 monomer. We have interpreted these results as indicating that extensive internal homology exists among lysine- and methionine-containing peptides within the leucyl-tRNA synthetase. The simplest conclusion that can be drawn from these observations is that the NH 2 - and COOH-terminal halves of leucyl-tRNA synthetase and perhaps other synthetases of 100,000 molecular weight may have evolved through a process of gene duplication and fusion, followed by limited diversification by way of amino-acid substitutions accumulating during evolution.

Ligand binding stoichiometries, subunit structure, and slow transitions in aminoacyl-tRNA synthetases

“…1974). There is the further complication that the monomeric enzymes of molecular weight 110 000 have extensive regions of duplicated primary structure (Koch et al, 1974; Kula, 1973; Waterson and Konigsberg, 1974;Bruton, 1975). One possibility is that these monomers are functionally similar to the dimers of molecular weight about 2 X 50 000; that is, they are effectively "covalently linked" dimers.…”

Configurational Statistics of Polynucleotide Chains. An Updated Virtual Bond Model to Treat Effects of Base Stacking

“…The five combinations of angles noted in boldface entail base stacking and describe loosely wound helical backbones;3,25 moreover, four of these five stacked geometries fit known X-ray fiber diffraction patterns. 20 In contrast to the conventional concept of a completely unstacked random coil, approximately 5 out of 12, or 42%, of the nucleotides in the poly(rA) chain at high temperature assume stacked geometries typical of ordered systems. In other words, only 58% of the poly(rA) molecule unstacks in this helix-coil transition!…”

Structure of the subunits of transcarboxylase and their relationship to the quaternary structure of transcarboxylase

“…Peptide mapping has, in some cases, however, successfully demonstrated sequence homology that may have arisen by gene duplication and fusion. The following are examples: human transferrin (Mann et al, 1970), leucyl-tRNA synthetase (Waterson and Konigsberg, 1974), methionyl-tRNA synthetase (Koch et al, 1974;Bruton et al, 1974), and valyl-tRNA synthetase (Koch et al, 1974). With each of these proteins the peptide maps revealed fewer peptides than expected for a nonduplicated sequence.…”

“…Succinylation of the Carboxymethylated Proteins. The method of succinylation with a few minor changes was that of Waterson and Konigsberg (1974). Each of the proteins (1 mg) was dissolved in 0.3 M sodium phosphate, pH 9.0, containing 6.0 M guanidine hydrochloride and treated with 10 mg of succinic anhydride in portions of 2,2, 2, and 4 mg.…”