Recognition between tRNASer and archaeal seryl-tRNA synthetases monitored by suppression of bacterial amber mutations.
Two dissimilar seryl-tRNA synthetases (SerRSs) exist in Methanosarcina barkeri: one of bacterial type (bMbSerRS) and the other resembling SerRSs present only in methanogenic archaea (mMbSerRS). While the expression of the archaeal bMbSerRS gene in Escherichia coli complements the function of thermolabile SerRS at a nonpermissive temperature, mMbSerRS does not. Our recent X-ray structural analysis of mMbSerRS revealed an idiosyncratic N-terminal domain and a catalytic zinc ion in the active site, identifying methanogenic-type SerRSs as atypical members of the SerRS family. To shed further light on substrate discrimination by methanogenic-type SerRS, we developed an in vivo system in E. coli to study tRNA serylation by mMbSerRS variants. We show that coexpression of the M. barkeri SerRS gene, encoding either bacterial- or methanogenic-type SerRS, with the gene for cognate archaeal suppressor tRNA leads to suppression of bacterial amber mutations, implying that the E. coli translation machinery can use serylated tRNA from methanogenic archaea as a substrate in protein synthesis. Furthermore, because serylation of M. barkeri serine-specific tRNA by endogenous E. coli SerRS is negligible, suppression is entirely dependent on recognition between archaeal partners (mMbSerRS/suppressor tRNA(Ser)). Thus, the efficiency of suppression by mMbSerRS variants quantified in the described beta-galactosidase-based reporter system, accurately reflects enzymes' serylation propensity obtained by in vitro kinetic measurements.
Lesjak
Sonja
S
Department of Chemistry, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Weygand-Durasevic
Ivana
I
D018389:Codon, Nonsense; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D017021:Methanosarcina barkeri / Q000201:enzymology*; D008958:Models, Molecular; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism*; D012362:RNA, Transfer, Ser / Q000378:metabolism*; D012714:Serine-tRNA Ligase / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D018389:Codon, Nonsense; D012362:RNA, Transfer, Ser; D012343:RNA, Transfer; D001616:beta-Galactosidase; D012714:Serine-tRNA Ligase