Abstract
Seryl-tRNA synthetase (SerRS) from methanogenic archaeon Methanosarcina barkeri, contains an idiosyncratic N-terminal domain, composed of an antiparallel beta-sheet capped by a helical bundle, connected to the catalytic core by a short linker peptide. It is very different from the coiled-coil tRNA binding domain in bacterial-type SerRS. Because the crystal structure of the methanogenic-type SerRSxtRNA complex has not been obtained, a docking model was produced, which indicated that highly conserved helices H2 and H3 of the N-terminal domain may be important for recognition of the extra arm of tRNA(Ser). Based on structural information and the docking model, we have mutated various positions within the N-terminal region and probed their involvement in tRNA binding and serylation. Total loss of activity and inability of the R76A variant to form the complex with cognate tRNA identifies Arg(76) located in helix H2 as a crucial tRNA-interacting residue. Alteration of Lys(79) positioned in helix H2 and Arg(94) in the loop between helix H2 and beta-strand A4 have a pronounced effect on SerRSxtRNA(Ser) complex formation and dissociation constants (K(D)) determined by surface plasmon resonance. The replacement of residues Arg(38) (located in the loop between helix H1 and beta-strand A2), Lys(141) and Asn(142) (from H3), and Arg(143) (between H3 and H4) moderately affect both the serylation activity and the K(D) values. Furthermore, we have obtained a striking correlation between these results and in vivo effects of these mutations by quantifying the efficiency of suppression of bacterial amber mutations, after coexpression of the genes for M. barkeri suppressor tRNA(Ser) and a set of mMbSerRS variants in Escherichia coli.
Highlights
The aminoacyl-tRNA synthetases2 catalyze the activation of cognate amino acids and their transfer to the 3Ј-end of
In the model the long variable arm of the tRNA is positioned to interact with the N-terminal domain of mMbSerRS, in accordance with our previous biochemical experiments that identified the long variable arm of archaeal tRNASer as a major tRNA recognition determinant [27, 29, 32]
Effects of Engineered mMbSerRS Amino Acid Alterations on Serylation of tRNA in Vivo—We have recently shown that expression of the gene encoding M. barkeri bacterial-type Seryl-tRNA synthetase (SerRS) in E. coli complements the function of thermolabile SerRS at the nonpermissive temperature, whereas expression of the mMbSerRS gene does not [28]
Summary
Site-directed Mutagenesis and Purification of Proteins—The seryl-tRNA synthetase expression vector (pET15bmMbSerRS) has been reported previously [27]. The tRNA transcript was carefully renatured prior to use in the kinetic assay, gel mobility shift assay, and surface plasmon resonance spectroscopy by heating for 5 min at 70 °C in 10 mM Tris/HCl, pH 7.5, followed by addition of MgCl2 to the final concentration of 5 mM and placing on ice. MbtRNAGSeGr A was maximally serylated to 80% as determined in the standard reaction mixture (described below). Electrophoretic Mobility Shift Assay—To check for complex formation between cognate tRNA and wild type or mutated mMbSerRS a constant amount of purified protein (8.3 pmol) was mixed with tRNA (14.8 pmol) and incubated for 15 min at 37 °C in a 13.5-l volume containing 30 mM Hepes, pH 7.0, and 6 mM MgCl2 followed by cooling on ice. To test salt influence on non-covalent complex formation between protein and nucleic acid, salt (25 or 250 mM KCl) at different amounts was added in the reaction mixture prior to incubation for 15 min at 37 °C. The efficiency of suppression was determined by measuring the -galactosidase activity produced from lacI-lacZ fusion harboring a nonsense mutation in the lacI portion [31]
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