Abstract
The yeast nuclear gene RML2, identified through genomic sequencing of Saccharomyces cerevisiae chromosome V, was shown to encode a mitochondrial homologue of the bacterial ribosomal protein L2. Immunoblot analysis showed that the mature Rml2p is a 37-kDa polypeptide component of the mitochondrial 54 S large ribosomal subunit. Null mutants of RML2 are respiration-deficient and convert to [rho-] or [rho degrees ] cytoplasmic petites, indicating that Rml2p is essential for mitochondrial translation. RML2 is regulated transcriptionally in response to carbon source and the accumulation of Rml2p is dependent on the presence of the 21 S large rRNA. Site-directed mutagenesis showed that a highly conserved 7-amino acid sequence (Val336 to Asp342) of Rml2p is essential for function. Substitution of Gln for His-343, the most highly conserved histidine in the L2 protein family, caused cold-sensitive respiratory growth but did not affect the assembly of 54 S ribosomal subunits. Mitochondrial protein synthesis was normal in the His343 to Gln (H343Q) mutant grown at the permissive temperature (30 degrees C) and was severely impaired after growth at the nonpermissive temperature (18 degrees C). His343 corresponds to His229 in Escherichia coli L2, which has been implicated in a direct involvement in peptidyl transferase activity. The conditional phenotype of the H343Q mutant indicates that His343 is not essential for peptidyl transferase activity in yeast mitochondria.
Highlights
Members of the L2 family of ribosomal proteins are highly conserved and are found in eubacteria, archaebacteria and in the cytoplasm and organelles of eukaryotes [1, 2]
Chemical modification of the histidine residues in the Escherichia coli and Bacillus stearothermophilus L2 proteins affected the assembly of the 50 S subunit and strongly inhibited peptidyl transferase activity [7, 8], and the imidazole functional group of histidine has been proposed to participate in peptidyl transferase through general acid-base catalysis analogous to the catalytic mechanism of serine proteases (9 – 11)
The functional importance of the L2-binding region in the rRNA is suggested by the observation that a point mutation of U1696 to A in the yeast mitochondrial rRNA, which corresponds to U1796 of E. coli 23 S rRNA, caused cold-sensitive growth on nonfermentable carbon sources and reduced amounts of assembled large ribosomal subunits [20]
Summary
Members of the L2 family of ribosomal proteins are highly conserved and are found in eubacteria, archaebacteria and in the cytoplasm and organelles of eukaryotes [1, 2]. The L2 variants included a single substitution of His229 to Gln, a 7-amino acid deletion ⌬Thr222 to Asp228, and the 2 amino acid deletions ⌬Gly221 to Thr222 and ⌬Asp228 to His229 When these mutant proteins were overexpressed from a plasmid in the background of the normal chromosomal L2 gene, the cells could not grow at 37 °C, and sucrose gradient centrifugation of ribosomal particles from cells grown at 30 °C showed that all of the mutants accumulated abnormal 40 S particles in addition to the normal 50 S subunit. The 50 S subunits containing H229Q-L2 were inactive in peptidyl transferase activity These results support the possibility that His229 is an essential part of the peptidyl transferase catalytic center
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