Related Experiment Videos
Sequence divergence of seryl-tRNA synthetases in archaea
H S Kim1, U C Vothknecht, R Hedderich
1Department of Molecular Biophysics and Biochemistry, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.
Journal of Bacteriology
|December 16, 1998
Summary
Methanogenic archaea form cysteinyl-tRNA (Cys-tRNACys) directly, not through serine misacylation. Seryl-tRNA synthetase (SerRS) enzymes from these organisms do not mischarge tRNACys with serine.
Area of Science:
- Molecular Biology
- Biochemistry
- Archaea Genomics
Background:
- Methanogenic archaea possess unique seryl-tRNA synthetase (SerRS) sequences and lack canonical cysteinyl-tRNA synthetase (CysRS).
- This raises questions about the mechanism of Cys-tRNACys formation: direct aminoacylation or serine misacylation followed by transformation.
Purpose of the Study:
- To investigate the enzymatic properties of SerRS from methanogenic archaea.
- To determine whether SerRS mischarges tRNACys with serine in these organisms.
Main Methods:
- Purification of SerRS from Methanobacterium thermoautotrophicum.
- Expression of SerRS from a cloned Methanococcus maripaludis serS gene.
- Enzymatic assays measuring aminoacylation of homologous and heterologous tRNAs.
- Gel shift experiments to assess SerRS interaction with tRNACys.
Main Results:
- Purified and expressed SerRS enzymes charged serine to their cognate tRNAs.
- These SerRS enzymes also accepted Escherichia coli tRNA as a substrate.
- Crucially, gel shift experiments demonstrated that M. thermoautotrophicum SerRS did not mischarge tRNACys with serine.
Conclusions:
- Cys-tRNACys is formed by direct aminoacylation in the methanogenic archaea studied.
- The uncommon SerRS sequence in these organisms does not lead to mischarging of tRNACys with serine.