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.

We have developed a system to transcribe the yeast 5S rRNA gene in the absence of the transcription factor TFIIIA. A TFIIIA is one of the most extensively studied and, hence, characterized transcription factors, for several reasons. First, it has the striking property of binding both 5S DNA and 5S RNA.Furthermore, its study led to the discovery of the zinc-finger motifs, which are structural units found only in eukaryotes and which are responsible for DNA binding (2, 3). These properties, common to all TFIIIA proteins studied so far, were first identified inXenopus TFIIIA, whose study was facilitated by its presence in large amount in oocytes. Xenopus TFIIIA possesses nine zinc fingers with specialized functions. The three amino-terminal fingers are primarily responsible for DNA binding, whereas fingers 4-7 are involved in RNA binding. The three carboxyl-terminal fingers and a 14-amino acid sequence in the carboxyl region of TFIIIA are required for transcriptional activation (4-6). Multiple activities have been attributed to Xenopus TFIIIA, including a role in DNA relaxation, double-stranded DNA reassociation (7,8), and being a weak DNA-dependent ATPase (9). These activities could be directly relevant to the mechanism of gene activation. TFIIIA could also have a regulatory role. In Xenopus, in Vitro transcription complexes preassembled on 5S genes can prevent nucleosomal repression (10). In vivo, the relative equilibrium between the amount of TFIIIA and histone Hi is, at least for oocyte 5S genes, very likely a major determinant of the activation (11,12). Recently, it was also shown in a human homologous in vitro system that binding of TFIIIA could prevent nucleosomal repression (13). TFIIIA can also exert a negative-feedback regulation through its binding to 5S RNA. Indeed, it was shown that 5S rRNA inhibits its own synthesis by sequestering TFIIIA (14)(15)(16) (24). Plasmid pRS-RPR1-5S is derived from the multicopy plasmid pRS424-5'3'RPR described by Pagan-Ramos et al. (25), which contains the 5' and 3' sequences flanking the mature domain of the RPR1 gene, together with TRPI as the selectable marker. pRS424-5'3'RPR was linearized by digestion with EcoRI, which cuts at the end of the 84-bp RPR1 leader promoter sequence, blunt-ended by digestion with mung bean nuclease, dephosphorylated, and ligated with a DNA fragment containing the yeast 5S DNA sequence, which was amplified by PCR from the pBS-5S plasmid. The primer at the 5' end of 5S DNA used for the amplification was 18 nt long and began at nt +2 (the second G) of the 5S DNA. The sequence at the junction between RPR1 and 5S DNA is GATTGGCAG*GTTGCG; the G* represents G + 1 of the 5S DNA, and the underlined sequence corresponds to the upstream part of the 5' primer.Abbreviations: ICR, internal control region; Pol III, RNA polymerase III; 5-FOA, 5-fluoroorotic acid. tTo whom reprint requests should be addressed.