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D R Engelke

Publications and source records attributed to D R Engelke.

At least 37 records · Page 2Linked to original sources
PubMed1996

An RNase P RNA subunit mutation affects ribosomal RNA processing.

RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed. This abundant RNA was identified as a 3'extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA. The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.

Base Sequence↗
PubMed1996

Mutational analysis of Saccharomyces cerevisiae nuclear RNase P: randomization of universally conserved positions in the RNA subunit.

Three regions in the Saccharomyces cerevisiae RNase P RNA have been identified, at positions Sce 87-94, Sce 309-316, and Sce 339-349, that contain nucleotides that are invariant in identity and position among all the known RNase P RNAs. To study the importance of these conserved RPR1 RNA regions in enzyme function, three independent mutational libraries were created in which the positions of invariant nucleotides were randomized simultaneously. Screening in vivo was used to identify viable RPR1 variants when reconstituted into holoenzyme in cells. Despite the universal evolutionary conservation, most of these positions tolerate certain sequence changes without severely affecting function. Most changes, however, produced subtle defects in cell growth and RNase P function, supporting the importance of these conserved regions. Isolation of conditional growth mutants allowed the characterization of the effects of mutations on cell growth, RPR1 RNA maturation, and activity of the holoenzyme in vitro. Kinetic analysis showed that viable variants were usually more defective in catalytic rate (Kcat) than in substrate recognition (Km).

Base Sequence↗
PubMed1996

A conserved RNA motif involved in divalent cation utilization by nuclear RNase P.

Catalytic RNAs are metalloenzymes that require precise coordination of divalent cation cofactors. In RNase P RNA, a conserved structural subdomain that has been implicated in magnesium coordination contains the consensus sequence acAGaRA. Randomization mutagenesis of the analogous sequence in the Saccharomyces cerevisiae nuclear RNase P RNA gene, RPR1, gave viable sequence variants that confer magnesium-correctable growth defects and are defective in magnesium cofactor utilization by the RNase P holoenzyme in vitro. Kinetic analysis of the defective holoenzymes suggests that the primary effects were on catalytic rate, rather than substrate recognition. The possible involvement of this RNA subdomain in catalysis is discussed.

Base Sequence↗
PubMed1995

Analysis of conserved positions in nuclear RNase P RNA.

Secondary structure models of eubacterial and eukaryotic nuclear RNase P RNA subunits show extensive structural similarities, allowing the identification of highly conserved nucleotide positions and molecular modeling of the enzyme-substrate complex in three dimensions. Based on this information, we present a preliminary tertiary structure model of the yeast nuclear RNase P RNA. In addition, the most conserved positions in the structure have been subjected to sequence randomization, with viable sequence variations identified by selection in vivo and characterized for phenotypic consequences.

Base Sequence↗
PubMed1994

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.

Base Sequence↗
PubMed1994

Structure-sensitive RNA footprinting of yeast nuclear ribonuclease P.

Several enzymatic and chemical reagents were used to probe the secondary structure of Saccharomyces cerevisiae nuclear RNase P RNA in the presence and absence of its protein components. Double-stranded regions were detected with RNase V1 and single-stranded regions with RNase ONE (Escherichia coli RNase I). Nucleotides not paired at Watson-Crick positions were monitored with dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-[2-(N-methylmorpholinio)ethyl]carbodiimide p-toluenesulfonate. The results supported most aspects of the previously proposed, phylogenetically-derived RNA secondary structure, although minor refinements allowed incorporation of both the biochemical and phylogenetic data. Digestion of the RNase P protein(s) with proteinase K gave enhanced reactivities to structure probes at selected positions, indicating regions of the RNA made inaccessible by the presence of the protein subunit(s). The regions of RNA protected in the yeast nuclear holoenzyme were considerably more extensive than that seen in the Escherichia coli holoenzyme, consistent with the observation that the protein moiety generally comprises a larger percentage of the RNase P holoenzyme in eukaryotes than in eubacteria.

Base Composition↗
PubMed1994

Replacement of the Saccharomyces cerevisiae RPR1 gene with heterologous RNase P RNA genes.

Phylogenetic studies of yeast nuclear RNase P RNA genes have shown a striking conservation of secondary structure for the Saccharomyces and Schizosaccharomyces RNase P RNAs, yet much of the primary sequence and many substructures vary among the RNAs examined. To investigate which sequences and structural features can be varied and still allow function in a heterologous organism, RNase P genes from several yeast species were tested for the ability to substitute for the Saccharomyces cerevisiae RNA. The RNase P genes from Saccharomyces carlsbergensis and Saccharomyces kluyveri could act as the sole source of RNase P RNA within S. cerevisiae cells, whereas the genes from Saccharomyces globosus and Schizosaccharomyces pombe could not. Although heterologous RNase P RNAs were synthesized by the cells in all cases, the RNAs that complemented tended to be processed from longer precursor transcripts into mature-sized RNase P RNA, while the RNAs that did not complement tended to accumulate as the longer precursor form. The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes.

Base Sequence↗
PubMed1994

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.

Base Sequence↗
PubMed1993

Gal4 protein binding is required but not sufficient for derepression and induction of GAL2 expression.

The Saccharomyces cerevisiae GAL2 gene upstream activator sequence (UAS) region was examined for protein bound in vivo by chromatin footprinting at high resolution. Gal4 transcriptional activator protein binds to the two consensus UAS sites whether GAL2 expression is induced, uninduced, or repressed by growth with different carbon sources. Although wild type strains show loss of the Gal4 protein-specific footprint in repressing media containing glucose, constitutive high level expression of Gal4 protein restores the GAL2 UAS footprints without fully derepressing GAL2 transcription. Thus binding of the Gal4 activator to target sites in the DNA is required but not sufficient for GAL2 derepression and induction. Gal4-independent protein-DNA complexes were also detected in the region, including one over the previously noted centromere-binding protein (CP1) site upstream of the Gal4 complexes.

Base Sequence↗
PubMed1993

Comparative structural analysis of nuclear RNase P RNAs from yeast.

Secondary structure models for yeast nuclear RNase P RNAs were derived by phylogenetic comparative analysis. RNase P RNA genes from six Saccharomyces species were characterized and compared with the published gene sequences of Saccharomyces cerevisiae (RPR1), Schizosaccharomyces pombe, and Schizosaccharomyces octosporus. The general organization of the Saccharomyces genes were similar: all were present in single copy and contained RNA polymerase III-specific regulatory elements, including tRNA gene-like A- and B-box promoters located within 5' leader regions and poly(T) terminators following the mature RNA domain. As observed previously, two RNase P RNAs were present in each of the species: a shorter RNA corresponding to the mature domain and a longer possible precursor RNA that includes the 5' leader sequences. The mature RNA domains of three of these genes were sufficiently divergent from the S. cerevisiae RNA such that compensatory base changes in paired elements were readily identified, yet homologous regions could be aligned. A striking common core of primary and secondary structure emerged for the Saccharomyces RNase P RNAs. Furthermore, the Schizosaccharomyces homologs conformed in large part to the Saccharomyces conserved core and shared with it a distinctive structural domain that has so far only been observed in the yeast nuclear RNase P RNAs. Comparison of the yeast core to a previously published eubacterial conserved core and to the RNA homologs from vertebrates revealed a number of similarities, suggesting that RNase P RNA from diverse sources may share a core of structurally conserved elements.

Base Sequence↗
PubMed1991

Expression of RNase P RNA in Saccharomyces cerevisiae is controlled by an unusual RNA polymerase III promoter.

The RNA subunit of Saccharomyces cerevisiae nuclear RNase P is encoded by a single-copy, essential gene, RPR1. The 369-nucleotide mature form of the RNA has an apparent precursor with an 84-nucleotide 5' leader and approximately 33 nucleotides of additional 3' sequence. Analysis of RPR1 transcription in a strain with a temperature-sensitive lesion in RNA polymerase III shows that the gene is transcribed in vivo by RNA polymerase III. Examination of potential promoter regions using both progressive upstream deletions and point mutations indicates that at least two sequences contained within the 5' leader region are essential for expression in vivo, while sequences farther upstream influence efficiency. The required leader elements resemble tRNA gene-like A-box and B-box internal promoters in sequence and spacing. As in the tRNA genes, transcription factor TFIIIC binds to this region in vitro and binding is severely reduced by either A-box or B-box point mutations that impair expression in vivo. It thus appears that the yeast RNase P RNA gene has adopted a promoter strategy that places an RNA polymerase III "internal" promoter upstream of the mature structural domain to help drive transcription.

Base Sequence↗
PubMed1991

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.

DNA↗
PubMed1991

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.

Base Sequence↗
PubMed1990

Chromosomal footprinting of transcriptionally active and inactive oocyte-type 5S RNA genes of Xenopus laevis.

The chromatin structure of the Xenopus oocyte-specific 5S rRNA genes was examined at high resolution in immature oocyte and somatic cell chromosomes by DNase I footprinting. On oocyte chromatin, where the genes are active, the cleavage preferences over the entire gene region showed a periodic pattern of sensitivity and were dramatically different from the patterns obtained with deproteinized DNA or somatic cell chromatin. Further, the normal binding site for TFIIIA over the internal promoter region was preferentially sensitive to cleavage, indicating that TFIIIA was not bound in the manner predicted by in vitro experiments. In somatic cell chromatin, the oocyte-type 5S genes displayed a cleavage pattern largely similar to deproteinized DNA suggesting the absence of positioned nucleosomes on these inactive genes, although the presence of uncharacterized repressor complexes could not be ruled out. These data are discussed in terms of potential forms of the chromatin structure and alternative mechanisms of oocyte-type gene activation.

Animals↗