The yeast homolog of mammalian ribosomal protein S30 is expressed from a duplicated gene without a ubiquitin-like protein fusion sequence. Evolutionary implications.
In mammals, the 59-residue ribosomal protein S30 (rpS30) is synthesized as a fusion to a 74-residue ubiquitin-like protein, which is cleaved to yield mature rpS30. An artificial fusion of this ubiquitin-like protein to E. coli beta-galactosidase was not cleaved when expressed in yeast (Saccharomyces cerevisiae), indicating that yeast lack this cleaving activity. The yeast rpS30 homolog (yrpS30) was purified and sequenced to reveal a 63-residue protein with 61% sequence identity to mammalian rpS30. Degenerate oligonucleotides based on the yrpS30 sequence were used to isolate full-length yrpS30 cDNAs. Sequence analysis of five cDNA clones revealed that yrpS30 is not synthesized as a fusion to a ubiquitin-like protein but is extended at its N terminus by a single methionine residue. The corresponding gene was identified in the GenBankTM data base by sequence alignment and termed RPS30A. The gene consists of two exons separated by a 430-base pair intron, which contains consensus splicing elements. Exon 1 encodes the initiator methionine residue and is preceded by canonical yeast ribosomal protein gene promoter elements. Exon 2 encodes the 62-residue mature yrpS30. Genomic hybridization reveals that the RPS30A gene is duplicated. Disruption of the RPS30A gene is not lethal but confers a slow growth phenotype. Ribosomes in the mutant strains contain an authentic yrpS30 protein, indicating that a functional yrpS30 is expressed from the duplicated gene but that the reduced capacity for yrpS30 synthesis restricted the growth rate. Analysis of available DNA sequence data bases reveals that rpS30 is synthesized as a fusion to a ubiquitin-like protein in nematodes and mammals but unfused in yeast, plants, and protazoa.
Baker
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RT
Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, Australian Capital Territory 2601, Australia.
Williamson
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NA
Wettenhall
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RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D008322:Mammals; D008969:Molecular Sequence Data; D005810:Multigene Family; D051381:Rats; D012269:Ribosomal Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014452:Ubiquitins / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D012269:Ribosomal Proteins; D014452:Ubiquitins; C040469:ribosomal protein S30; D001616:beta-Galactosidase; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase