The only essential function of TFIIIA in yeast is the transcription of 5S rRNA genes.

Abstract

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.

A Hydrophobic Segment within the 81-Amino-Acid Domain of TFIIIA from Saccharomyces cerevisiae Is Essential for Its Transcription Factor Activity

“…Therefore, as an alternative approach for monitoring the activity of TFIIIA, we tested the ability of these mutant forms of TFIIIA to support cell growth. We note that the only essential function of TFIIIA in vivo is in promoting transcription of the 5S RNA gene (12).…”

Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

“…Even though it is believed that fungal and mammalian mitochondrial ribosomes do not contain 5S rRNA, it is not clear whether ribosomes in general can perform translation in its absence (Camier et al, 1995). Clearly, further work is required to elucidate the role of this small rRNA in mammalian mitochondria.…”

The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships

“…We performed separate ChIP microarray experiments with two members of each of the basal transcription complexes: Pol III (Rpc82 and Rpc40), TFIIIB (Brf1 and TBP), and TFIIIC (Tfc1 and Tfc6). TFIIIA was not included as it is reported to have a single essential target, 5S rDNA (24). Input and ChIP-enriched DNA were labeled (Cy3 for input, Cy5 for ChIPenriched) and hybridized competitively to glass slides containing the entire yeast genome arrayed in Ϸ14,000 segments, parsed as ORFs and intergenic regions (see Supporting Materials and Methods).…”