STP1, a gene involved in pre-tRNA processing, encodes a nuclear protein containing zinc finger motifs.

Abstract

STPI is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STPI might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STPI gene and its products. The STPI locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STPI open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STPI ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STPI ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing.The genes encoding tRNAs can contain intervening sequences (IVS). In Saccharomyces cerevisiae, 10 families of tRNA contain IVS (66; for a review, see reference 9). The steps involved in removing IVS from pre-tRNAs have been the subject of numerous studies and are currently well described (for a review, see reference 9). The first step is endonucleolytic cleavage to remove the IVS. Cleavage is catalyzed by tRNA-splicing endonuclease, generating 5' half molecules containing 2',3' cyclic phosphates and 3' half molecules with 5' hydroxyl groups (51). The tRNA splicing endonuclease is composed of three dissimilar subunits (54), one of which is probably encoded by the essential SEN2 gene (23). The cognate half molecules are ligated in a second step by tRNA ligase, which catalyzes the phosphorylation and activation of the 3' half as well as the joining step (19,52). The ligase is a single-subunit enzyme encoded by the essential RLGI gene (52,53,71). The ligated molecule contains both 3' and 2' phosphates at the splice junction. The 2' phosphate is removed in the final step by a 2' tRNA phosphotransferase activity (44,45). The gene encoding this activity has not been identified. Thus, of the five peptides known to be required for pre-tRNA splicing in vitro, the genes encoding two, SEN2 and RLGI, have been characterized.There are a large number of yeast genes that when mutated result in inefficient removal of IVS from pre-tRNAs. These include RNAI (25,35) reaction (27). LOSI and STPI are unessential genes whose functions are also unknown.STPI was identified by searching for wild-type genes that when overexpressed would ...

Receptor-mediated endoproteolytic activation of two transcription factors in yeast

“…The Stp1p expressed from this construct exhibited an identical pattern of migration as the protein expressed from the endogenous promoter, and also displayed the same amino-acid-dependent shift in mobility (data not shown). These data confirm that STP1 translation normally initiates at the second start codon, a result that indicates that the transcriptional start site, originally mapped by Wang et al (1992), is not influenced by the presence of inducing amino acids.…”

“…1A). Plasmids pCA022 and pCA024 were found to contain identical inserts, and pCA023 contained a shorter fragment derived from the same chromosomal region surrounding the STP1 locus (Wang et al 1992).…”

“…In the original characterization of STP1, it was found that transcription initiates within and not upstream of the STP1 ORF (Wang et al 1992), at a position between the first and second ATG codons (see Fig. 1B).…”

A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome

“…Examples include binding sites for Yap7, which belongs to a class of bZIP stress response regulators (Fernandes et al 1997), and the zinc-finger transcription factor Stp1 ( Fig. 3C) (Wang et al 1992).…”

Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain

“…Beier. Wang et al, 1992). The endonuclease apparently recognizes features that are shared by all of its substrates; (a) the IVS is always located one nucleotide 3' to the anticodon (Ogden et al, 1984), (b) the 3' splice site is present in a single-stranded loop and the 5'junction is located either in a single-stranded or labile double-stranded region, (c) the precursors probably form a tRNA-like tertiary structure in the mature domain of the molecule as suggested by chemical and enzymic structurespecific probes (Lee and Knapp, 1985;Swerdlow and Guthrie, 1984).…”