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Misacylation of pyrrolysine tRNA in vitro and in vivo

Sarath Gundllapalli1, Alexandre Ambrogelly, Takuya Umehara

  • 1Departments of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.

FEBS Letters
|September 9, 2008
PubMed

Insights

Methanosarcina barkeri pyrrolysine (Pyl) tRNA can be mistakenly charged with serine by a bacterial seryl-tRNA synthetase. Specific mutations enable this tRNA to be charged by E. coli alanyl-tRNA synthetase.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Methanosarcina barkeri utilizes pyrrolysine (Pyl) for protein synthesis, inserting it at UAG codons.
  • Pyrrolysyl-tRNA synthetase (PRS) is responsible for attaching Pyl to its cognate tRNA (tRNAPyl).

Purpose of the Study:

  • To investigate the substrate specificity of M. barkeri tRNAPyl.
  • To determine if M. barkeri tRNAPyl can be misacylated by other aminoacyl-tRNA synthetases.
  • To identify sequence elements in M. barkeri tRNAPyl responsible for its recognition by synthetases.

Main Methods:

  • In vitro aminoacylation assays using purified synthetases and tRNAs.
  • In vivo experiments in Escherichia coli to assess tRNA misacylation.
  • Site-directed mutagenesis of M. barkeri tRNAPyl.

Main Results:

  • M. barkeri Fusaro tRNAPyl was misacylated with serine by M. barkeri seryl-tRNA synthetase in vitro and in vivo.
  • M. barkeri MS tRNAPyl contains a G3:U70 pair, a known identity element for E. coli alanyl-tRNA synthetase (AlaRS).
  • Despite the G3:U70 pair, M. barkeri MS tRNAPyl was not aminoacylated by E. coli AlaRS, but a mutation at the A4:U69 pair enabled AlaRS recognition.

Conclusions:

  • M. barkeri tRNAPyl can be mischarged by a bacterial seryl-tRNA synthetase, highlighting potential cross-reactivity.
  • Specific nucleotide pairs in the tRNA acceptor stem dictate recognition by different aminoacyl-tRNA synthetases.
  • Understanding tRNA-synthetase interactions is crucial for the fidelity of the genetic code.

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