Cloning and analysis of the nuclear gene for YmL33, a protein of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae

Abstract

The N-terminal amino acid sequence of a large subunit protein, termed YmL33, of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae was determined. The data were obtained to synthesize two kinds of oligonucleotide primers, which were used in the polymerase chain reaction to amplify and clone the nuclear gene for this protein. By nucleotide sequencing, the cloned gene, MRP-L33, was found to encode a basic protein of 11 kDa with 98 amino acid residues. The protein encoded by this gene appears to have no leader sequence at its N terminus. The N-terminal two-thirds of the deduced amino acid sequence showed a significant degree of sequence similarity to ribosomal protein L30 of Escherichia coli and Bacillus stearothermophilus. In addition, the C-terminal one-third showed sequence similarity, though to a lesser extent, to a yeast cytoplasmic ribosomal protein termed L16. By hybridization with the yeast chromosomes and their restriction enzyme fragments, the MRP-L33 gene was concluded to exist on chromosome XIII as a single-copy gene. Disruption of the gene by insertion of a HIS3-containing fragment showed that MRP-L33 was essential for mitochondrial function. The transcriptional level of MRP-L33 in strains with different mitochondrial genetic backgrounds was analyzed in the presence of glucose, galactose, or glycerol.