Isolation of two cDNAs encoding functional human cytoplasmic cysteinyl-tRNA synthetase

Area of Science:

• Molecular Biology
• Enzymology
• Genetics

Background:

• Eukaryotic cysteinyl-tRNA synthetase sequences possess insertions absent in bacterial counterparts.
• Previous studies on E. coli cysteinyl-tRNA synthetase provided comparative insights.
• A previously described human gene sequence (638 amino acids) showed carboxyl-terminal homology issues.

Purpose of the Study:

• To clone and express the full-length human cytoplasmic cysteinyl-tRNA synthetase gene.
• To determine the definitive sequence of the human enzyme.
• To investigate differences between bacterial and eukaryotic cysteinyl-tRNA synthetases.

Main Methods:

• Isolation of full-length cDNA encoding human cytoplasmic cysteinyl-tRNA synthetase.
• Sequence analysis of the isolated cDNA and predicted protein.
• Investigation of mRNA alternative splicing using RT-PCR or similar techniques.
• Expression of full-length and alternative enzyme forms in E. coli.

Main Results:

• A full-length cDNA encoding a 748-amino acid protein was isolated.
• The new sequence exhibits significant homology with other eukaryotic cysteinyl-tRNA synthetases.
• Alternative splicing in 20% of mRNA results in an 8-base insertion, altering the C-terminus.
• Expressed proteins, both full-length and alternative forms, were functional and active in aminoacylation.

Conclusions:

• The definitive sequence for human cytoplasmic cysteinyl-tRNA synthetase is 748 amino acids.
• Alternative splicing generates functional enzyme variants with altered C-termini.
• These findings elucidate key differences between bacterial and eukaryotic cysteinyl-tRNA synthetases.