The gene for MRP7, a 40-kilodalton protein of the large subunit of the yeast mitochondrial ribosome, was identified in a Agtll expression library by immunological screening with a monoclonal antibody to MRP7. An intact copy of MRP7 was then isolated from a yeast genomic library by colony hybridization. Gene disruption showed that MRP7 protein was essential for ribosomal function. Sequencing of MRP7 revealed a coding region for a basic (pI 10.6), 43.2-kilodalton protein containing 371 amino acid residues. Amino acid residues 28 to 112 of the deduced MRP7 sequence aligned with the 84 residues of the Escherichia coli ribosomal protein L27, but no significant similarity was detected between the carboxy-terminal 259 amino acids of MRP7 and other protein sequences in existing computer data bases. Within the aligned region, there was 49% amino acid identity between MRP7 and L27, compared with the 57% identity observed between L27 and its homolog in Bacilus stearothermophilus. The steady-state levels of the MRP7 protein and its mRNA were monitored in response to catabolite repression and to increased dosage of the MRP7 gene. The response to catabolite repression was characterized by a ninefold change in the level of the protein and little, if any, change in the level of the mRNA. In cells carrying the MRP7 gene on a high-copy-number plasmid, the mRNA was increased 20-fold, but there was no significant increase in MRP7 protein. Furthermore, MRP7 mRNA and protein accumulated at normal levels in [rho°] cells, which are devoid of 21S rRNA, indicating that the protein is relatively stable in the absence of ribosome assembly. Together, these results suggest that MRP7 is regulated posttranscriptionally, probably at the level of protein synthesis rather than protein turnover.Ribosomes are universal features of procaryotic and eucaryotic cells, and intensive investigation for over two decades has led to major advances in our understanding of their structure, function, synthesis, and evolution (for reviews, see references 16, 28, 31, 40, 44, and 66). Much of this knowledge has been developed in studies of the Escherichia coli ribosome, which has become the standard of comparison for the analysis of ribosomes in other organisms. In contrast to bacteria, eucaryotic cells maintain multiple, distinctly different ribosomal populations for protein synthesis in the cytoplasm, chloroplasts, and mitochondria. The presence of cytoplasmic and organellar ribosomes provides an opportunity to compare the properties of genetically separate sets of ribosomal components within a single cell and to establish relationships between these components and those of the E. coli ribosome. These comparisons should help to identify features of eucaryotic ribosomes that are special adaptations to the complex genomic organization and intracellular compartmentalization of eucaryotic cells. The ribosomes of mitochondria and chloroplasts are of special interest in this regard because the genes for the constituents of organellar ribosomes are divided between two...