RESUMO
A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.
Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico 5S/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Fúngicos , Cinética , Peso Molecular , Mutação , Conformação de Ácido Nucleico , Precursores de RNA/química , Precursores de RNA/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
We have developed a system to transcribe the yeast 5S rRNA gene in the absence of the transcription factor TFIIIA. A long transcript was synthesized both in vitro and in vivo from a hybrid gene in which the tRNA-like promoter sequence of the RPR1 gene was fused to the yeast 5S RNA gene. No internal initiation directed by the endogenous 5S rDNA promoter or any processing of the hybrid transcript was observed in vitro. Yeast cells devoid of transcription factor TFIIIA, which, therefore, could not synthesize any 5S rRNA from the endogenous chromosomal copies of 5S rDNA, could survive if they carried the hybrid RPR1-5S construct on a multicopy plasmid. In this case, the only source of 5S rRNA was the precursor RPR1-5S transcript that gave rise to two RNA species slightly larger than wild-type 5S rRNA. This establishes that the only essential function of TFIIIA is to promote the synthesis of 5S rRNA. However, cells devoid of TFIIIA and surviving with these two RNAs grew more slowly at 30 degrees C compared with wild-type cells and were thermosensitive at 37 degrees C.
