Abstract
The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).
Highlights
The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli
As already noted by Koonin [40], this group of hisT(truA)-like enzymes is rather distant from the pseudouridine synthases identified so far, specific, respectively, for ⌿55 (E. coli truB-like), for ⌿32 in tRNA and ⌿746 in 23 S rRNA (E. coli rluA-like), and for ⌿516 in E. coli 18 S rRNA
Yeast Protein Deg1 Is tRNA:⌿38/39 Synthase—Direct evidence that the yeast Deg1 protein is a tRNA:pseudouridine synthase comes from two types of experiments: by detecting the corresponding enzymatic activity in S100 extracts of a transformed E. coli strain harboring the yeast DEG1 gene and by measuring the enzymatic activity of a ProtA-Deg1 fusion protein purified from a deg1Ϫ yeast strain
Summary
Enzymes, and Materials—␣-32P-Radiolabeled nucleotide triphosphates (400 Ci/mmol) were from Amersham The DEG1 ORF was amplified by PCR from total yeast genomic DNA using two primers that created an XhoI restriction site at the ATG start codon (aaaaactcgagcAGTAATTTCATTAGAAGGCTAG) and an MluI restriction site (aaaaaacgcgtAAGAAATATAGTCTTCAAGG) in the 3Ј-untranslated region of the gene This manipulation allowed cloning of the ORF into a modified pET (pET-His6/pET8c) vector previously cut with XhoI/MluI and created an in-frame fusion protein of 6 histidine residues joined by a spacer Ser-Ser dipeptide to the amino acid immediately after the start methionine. Construction of the Deg1p Fusion Protein Carrying Protein A or Green Fluorescent Protein (GFP) as a Tag—Epitope tagging of Deg1p was done by fusing two IgG binding units from Staphyloccus aureus protein A to the N-terminal end of Deg1p For this gene fusion, a new PstI restriction site was generated at the ATG codon of DEG1 by PCR-mediated mutagenesis, and the ORF was subcloned into the plasmid pRS315 in-frame with the two IgG binding units under the control of the NOP1 promoter (PNop1-ProtA cassette; see Ref. 32), creating the plasmid pNOP-ProtA-Deg. Multiple sequence alignment was constructed using Macintosh version of MACAW software, version 2.0.5
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