Partial characterization of an RNA component that copurifies with Saccharomyces cerevisiae RNase P.

Saccharomyces cerevisiae cellular RNase P is composed of both protein and RNA components that are essential for activity. The isolated holoenzyme contains a highly structured RNA of 369 nucleotides that has extensive sequence similarities to the 286-nucleotide RNA associated with Schizosaccharomyces pombe RNase P but bears little resemblance to the analogous RNA sequences in procaryotes or S. cerevisiae mitochondria. Even so, the predicted secondary structure of S. cerevisiae RNA is strikingly similar to the bacterial phylogenetic consensus rather than to previously predicted structures of other eucaryotic RNase P RNAs.

Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

Comparison of tRNA gene transcription complexes formed in vitro and in nuclei.

The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C. Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNA(SUP53Leu) gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions. Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter. The chromosomal tRNA(SUP53Leu) and tRNA(UCGSer) genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.

ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences.

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5'-TC(T)4-6GCTG-3', which we propose constitutes an important part of the ACE1 consensus recognition sequence.

Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

tRNA genes as transcriptional repressor elements.

Eukaryotic genomes frequently contain large numbers of repetitive RNA polymerase III (pol III) promoter elements interspersed between and within RNA pol II transcription units, and in several instances a regulatory relationship between the two types of promoter has been postulated. In the budding yeast Saccharomyces cerevisiae, tRNA genes are the only known interspersed pol III promoter-containing repetitive elements, and we find that they strongly inhibit transcription from adjacent pol II promoters in vivo. This inhibition requires active transcription of the upstream tRNA gene but is independent of its orientation and appears not to involve simple steric blockage of the pol II upstream activator sites. Evidence is presented that different pol II promoters can be repressed by different tRNA genes placed upstream at varied distances in both orientations. To test whether this phenomenon functions in naturally occurring instances in which tRNA genes and pol II promoters are juxtaposed, we examined the sigma and Ty3 elements. This class of retrotransposons is always found integrated immediately upstream of different tRNA genes. Weakening tRNA gene transcription by means of a temperature-sensitive mutation in RNA pol III increases the pheromone-inducible expression of sigma and Ty3 elements up to 60-fold.

5' processing of tRNA precursors can Be modulated by the human La antigen phosphoprotein.

Eukaryotic precursor (pre)-tRNAs are processed at both ends prior to maturation. Pre-tRNAs and other nascent transcripts synthesized by RNA polymerase III are bound at their 3' ends at the sequence motif UUUOH [3' oligo(U)] by the La antigen, a conserved phosphoprotein whose role in RNA processing has been associated previously with 3'-end maturation only. We show that in addition to its role in tRNA 3'-end maturation, human La protein can also modulate 5' processing of pre-tRNAs. Both the La antigen's N-terminal RNA-binding domain and its C-terminal basic region are required for attenuation of pre-tRNA 5' processing. RNA binding and nuclease protection assays with a variety of pre-tRNA substrates and mutant La proteins indicate that 5' protection is a highly selective activity of La. This activity is dependent on 3' oligo(U) in the pre-tRNA for interaction with the N-terminal RNA binding domain of La and interaction of the C-terminal basic region of La with the 5' triphosphate end of nascent pre-tRNA. Phosphorylation of La is known to occur on serine 366, adjacent to the C-terminal basic region. We show that this modification interferes with the La antigen's ability to protect pre-tRNAiMet from 5' processing either by HeLa extract or purified RNase P but that it does not affect interaction with the 3' end of pre-tRNA. These findings provide the first evidence to indicate that tRNA 5'-end maturation may be regulated in eukaryotes. Implications of triphosphate recognition is discussed as is a role for La phosphoprotein in controlling transcriptional and posttranscriptional events in the biogenesis of polymerase III transcripts.

The human tRNA-modifying protein, TRIT1, forms amyloid fibers in vitro.

TRIT1 is a highly conserved tRNA isopentenyl transferase that modifies a subset of tRNAs in human cells and is a candidate tumor suppressor in lung cancer in certain ethnic populations. The yeast homologue, Mod5, has similar tRNA-modifying functions in the cytoplasm and is required for the transcriptional silencing activity of RNA polymerase II promoters near tRNA genes in the nucleus, a phenomenon termed tRNA gene mediated (tgm) silencing. Furthermore, Mod5 can fold into amyloid fibers in vitro and in vivo, which confers resistance to certain fungicides in yeast. Since TRIT1 complements both tRNA modifying and tgm-silencing activities in yeast where the Mod5 gene has been deleted, it seemed possible that TRIT1 might also have amyloid-forming capabilities. Here we show that TRIT1, like Mod5, directly binds to tRNAs that are both substrate and non-substrates for modification with similar affinity, and to an unstructured, non-tRNA. Binding appears to involve distinct protein-RNA multimers which decrease in electrophoretic mobility as the protein to RNA ratio increases. Furthermore, we characterize TRIT1 as a novel human amyloid fiber forming protein. We discuss these data in light of TRIT1's functional roles and possible implications for disease.

Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures.

Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via alpha-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.

The cytoplasmic and nuclear populations of the eukaryote tRNA-isopentenyl transferase have distinct functions with implications in human cancer.

Mod5 is the yeast tRNA isopentenyl transferase, an enzyme that is conserved from bacteria to humans. Mod5 is primarily cytoplasmic where it modifies the A37 position of a few tRNAs, and the yeast enzyme has been shown capable of forming heritable, amyloid-like aggregates that confer a selective advantage in the presence of specific antifungal agents. A subpopulation of Mod5 is also found associated with nuclear tRNA genes, where it contributes tRNA-gene mediated (tgm) silencing of local transcription by RNA polymerase II. The tgm-silencing function of Mod5 has been observed in yeast and a Mod5-deletion in yeast can be complemented by the plant and human tRNA isopentenyl transferases, but not the bacterial enzymes, possibly due to the lack of an extended C-terminal domain found in eukaryotes. In light of this additional nuclear role for Mod5 we discuss the proposed role of the human homologue of Mod5, TRIT1, as a tumor suppressor protein.

Interaction of tRNA transcription factors with satellite I DNA from Xenopus laevis.

A cloned repeat of Xenopus laevis satellite I DNA was tested for the ability to form stable complexes with tRNA transcription factors in vitro. In template exclusion studies, the satellite I DNA competed efficiently with a tRNA gene for binding of yeast RNA polymerase III transcription factors. DNase I footprinting further showed that transcription factor TF IIIC alone bound to satellite I DNA at both the A block and B block consensus promoter sequences immediately downstream from the transcription start point. The strength and position of these associations indicate that satellite I DNA is a potential site for association of the same DNA-binding proteins that activate tRNA gene transcription.

Yeast expression vectors using RNA polymerase III promoters.

A series of Saccharomyces cerevisiae--Escherichia coli shuttle vectors is described in which small RNAs can be stably expressed in yeast from two different promoters for RNA polymerase III transcription. The vectors are available in either high- or low-copy-number forms with either URA3, HIS3, or TRP1 selection markers, and are based on a previously described set of plasmid vectors [Sikorski and Hieter, Genetics 122 (1989) 19-27]. Transcripts have structured pre-tRNA or RPR1 leaders fused to RNA corresponding to inserted sequences. Levels of RNA accumulation are dependent on plasmid copy number and the type of transcript.

Direct identification of small sequence changes in chromosomal DNA.

Dideoxynucleotide chain termination sequencing has been applied directly to genomic DNA templates by annealing radiolabeled oligodeoxynucleotide primers to unique sites in total yeast DNA and extending with avian myoblastosis virus (AMV) reverse transcriptase. The technique is used here to confirm the introduction of selectively altered tRNA genes into the Saccharomyces cerevisiae genome by gene replacement.

Binding of yeast TFIIIC to tRNA gene bipartite internal promoters: analysis of physical effects on the intervening DNA.

Complexes between transcription factor TFIIIC and eukaryotic tRNA gene internal promoter A and B boxes are unusual in that the binding to the two distinct sites tolerates considerable variation in both distance and helical orientation between the sites. Electrophoretic mobility of Saccharomyces cerevisiae TFIIIC complexes with circularly permuted tRNA gene fragments and sensitivity of the complexes to a single stranded-specific reagent, potassium permanganate, indicated that no significant bend or distortion was introduced into the DNA by simultaneous binding to both internal promoters. These data support a model in which variability in the relative positions of the two binding sites is compensated by flexibility in the structure of TFIIIC.