Yeast ribosomal protein genes: I. Transcription factor binding. II. RPL30 structure and function

Abstract

I. There are at least 75 proteins in the Saccharomyces cerevisiae cytoplasmic ribosome. The synthesis of these proteins is coordinately regulated by the cell, primarily at the level of transcription. Previous deletion studies have characterized two consensus upstream sequences to be required for efficient transcription of most ribosomal protein genes. Using binding and protection analyses, it was determined that a trans-acting factor(s) binds specifically to CYH2, RPL30A-RPL32 and RPL30B UAS. Competition studies demonstrated that this binding factor has a higher affinity for some ribosomal protein gene upstream activating sequences (UAS) than for others and that this factor is distinct from two of the factors known to bind to the rRNA enhancer.;II. Many of the ribosomal proteins in S. cerevisiae are encoded by duplicate genes. Each ribosomal protein gene characterized thus far is transcribed, although to varying degrees. L30, a 60S subunit ribosomal protein, is encoded by two genes, RPL30A and RPL30B. Cloning and sequencing of RPL30B revealed a high level of homology to RPL30A in the coding region, with divergent 5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar} sequences. RPL30B contains an intron in its 5{dollar}\sp\prime{dollar} untranslated region, with a 3{dollar}\sp\prime{dollar} splice site in an analogous location to that of RPL30A, but with an unusual 5{dollar}\sp\prime{dollar} splice site, C/GUAUGU.;Genomic RPL30A and RPL30B were disrupted by homologous recombination. RPL30A is responsible for the majority of L30 production as demonstrated by growth rate and northern blot analyses of these disruption mutants. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yields four viable spores. Ribosome from haploid cells carrying both disrupted genes have no detectable L30. Yet such cells grow with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 does not alter the ratio of 60S:40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes and a decrease in 80S monosomes. Cross-linking experiments performed by Dr. John Lee (U. Texas, San Antonio) suggest that these mutant cells assemble their ribosomes differently.