Domain structure in yeast tRNA ligase

Yeast tRNA ligase is one of two proteins required for the splicing of precursor tRNA molecules containing introns. The 95-kDa tRNA ligase has been purified to homogeneity from a strain of Escherichia coli which overexpresses the protein. The ligation reaction requires three enzymatic activities: phosphodiesterase, polynucleotide kinase, and ligase. By partial proteolytic digestion, we have produced fragments of tRNA ligase which contain the constituent activities. These results provide evidence for a model in which the three constituent activities of ligase are located in three distinct domains separated by protease-sensitive regions. We have also located the active adenylylated site in the ligase domains. It is lysine-114. The tRNA ligase sequence in this region has limited homology to the active-site region of T4 RNA ligase.     

Structure-function analysis of yeast tRNA ligase

Trl 1 is an essential 827-amino-acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two catalytic domains--an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5'-kinase/cyclic phosphodiesterase component (amino acids 389-827)--that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery.     

A distinct subnuclear localization of mammalian DNA topoisomerase IIbeta in yeast

Mammalian topoisomerase II isoforms alpha and beta are diverged in their C-terminal domain (CTD), but both isoforms complement the yeast top2 mutation. In this study, mammalian topoisomerase IIalpha-CTD and IIbeta-CTD were tagged with yellow fluorescent protein (YFP), expressed in yeast cells, and their localization was examined. YFP tagged-topoisomerase IIalpha-CTD was distributed evenly throughout the nucleus, while YFP tagged-topoisomerase IIbeta-CTD was sequestered into a subnuclear compartment. Deletion analysis revealed that two regions (amino acids 1207-1234 and 1513-1573) of the topoisomerase IIbeta-CTD are essential for specific localization of the beta isoform: if either of the two regions is removed, the mutant topoisomerase IIbeta-CTD distributes evenly throughout the nucleus. The data suggest that yeast cells distinguish the nuclear and subnuclear localization signals associated with these two mammalian topoisomerase II isoforms.     

Mechanism of action of a yeast RNA ligase in tRNA splicing

The yeast endonuclease and ligase activities that carry out the splicing of tRNA precursors in vitro have been physically separated. The properties of a partially purified ligase fraction were examined. The ligase requires a divalent cation and a nucleoside triphosphate as cofactor. The product of ligation is a 2′-phosphomonoester, 3′,5′-phosphodiester linkage. The phosphate in the newly formed phosphodiester bond comes from the γ position of ATP, while the 2′ phosphate is derived from the RNA substrate. An adenylylated enzyme intermediate was identified by incorporation of label from α-³²P-ATP. Adenylylation was reversed by pyrophosphate, releasing ATP, whereas ligation was accompanied by release of AMP. Polynucleotide kinase and cyclic phosphodiesterase activities copurify with the adenylylated protein and may be required for the tRNA splicing reaction.     

Preferential binding of yeast tRNA ligase to pre-tRNA substrates.

Joining of tRNA halves during splicing in extracts of Saccharomyces cerevisiae requires each of the three enzymatic activities associated with the tRNA ligase polypeptide. Joining is most efficient for tRNA as opposed to oligonucleotide substrates and is sensitive to single base changes at a distance from splice sites suggesting considerable specificity. To examine the basis for this specificity, binding of ligase to labeled RNA substrates was measured by native gel electrophoresis. Ligase bound tRNA halves with an association constant 1600-fold greater than that for a nonspecific RNA. Comparison of binding of a series of tRNA processing intermediates revealed that tRNA-structure, particularly in the region around the splice sites, contributes to specific binding. Finally, the ligase was shown to form multiple, discrete complexes with tRNA substrates. The basis for recognition by ligase and its role in a tRNA processing pathway are discussed.     

Genetic and biochemical analysis of the functional domains of yeast tRNA ligase

Yeast tRNA ligase (Trl1) converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO4, 3'-5' phosphodiester at the splice junction. Trl1 performs three reactions: (i) the 2',3'-cyclic phosphate of the proximal fragment is hydrolyzed to a 3'-OH, 2'-PO4 by a cyclic phosphodiesterase (CPD); (ii) the 5'-OH of the distal fragment is phosphorylated by an NTP-dependent polynucleotide kinase; and (iii) the 3'-OH, 2'-PO4, and 5'-PO4 ends are sealed by an ATP-dependent RNA ligase. Trl1 consists of an N-terminal adenylyltransferase domain that resembles T4 RNA ligase 1, a central domain that resembles T4 polynucleotide kinase, and a C-terminal CPD domain that resembles the 2H phosphotransferase enzyme superfamily. Here we show that all three domains are essential in vivo, although they need not be linked in the same polypeptide. We identify five amino acids in the adenylyltransferase domain (Lys114, Glu266, Gly267, Lys284, and Lys286) that are essential for Trl1 activity and are located within motifs I (114KANG117), IV (266EGFVI270), and V (282FFKIK286) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligases 1 and 2. Mutations K404A and T405A in the P-loop (401GXGKT405) of the central kinase-like domain had no effect on Trl1 function in vivo. The K404A and T405A mutations eliminated ATP-dependent kinase activity but preserved GTP-dependent kinase activity. A double alanine mutant in the P-loop was lethal in vivo and abolished GTP-dependent kinase activity. These results suggest that GTP is the physiological substrate and that the Trl1 kinase has a single NTP binding site of which the P-loop is a component. Two other mutations in the central domain were lethal in vivo and either abolished (D425A) or severely reduced (R511A) GTP-dependent RNA kinase activity in vitro. Mutations of the signature histidines of the CPD domain were either lethal (H777A) or conferred a ts growth phenotype (H673A).     

Function of yeast and amphioxus tRNA ligase in IRE1alpha-dependent XBP1 mRNA splicing

During their maturation step, transfer RNAs (tRNAs) undergo excision of their introns by specific splicing. Although tRNA splicing is a molecular event observed in all domains of life, the machinery of the ligation reaction has diverged during evolution. Yeast tRNA ligase 1 (TRL1) is a multifunctional protein that alone catalyzes RNA ligation in tRNA splicing, whereas three molecules [RNA ligase (RNL), Clp1, and PNK/CPDase] are necessary for RNA ligation in tRNA splicing in amphioxi. RNA ligation not only occurs in tRNA splicing, but also in yeast HAC1 mRNA splicing and in animal X-box binding protein 1 (XBP1) mRNA splicing under conditions of endoplasmic reticulum (ER) stress. Yeast TRL1 is known to function as an RNA ligase for HAC1 mRNA splicing, whereas the RNA ligase for XBP1 mRNA splicing is unknown in animals. We examined whether yeast and amphioxus RNA ligases for tRNA splicing function in RNA ligation in mammalian XBP1 splicing. Both RNA ligases functioned in RNA ligation in mammalian XBP1 splicing in vitro. Interestingly, Clp1, and PNK/CPDase were not necessary for exon–exon ligation in XBP1 mRNA by amphioxus RNL. These results suggest that RNA ligase for tRNA splicing might therefore commonly function as an RNA ligase for XBP1 mRNA splicing.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

The methionine initiator tRNA genes of yeast

We have isolated three distinct tRNAimet genes from a yeast DNA clone bank. The complete sequence of two shows that these genes are colinear with the mature tRNAimet and supports the RNA sequence of tRNAimet. Southern analysis of yeast genomic DNA indicates the presence of four copies of tRNAimet gene per haploid genome.     

Predicting protein subnuclear localization using GO-amino-acid composition features

The nucleus guides life processes of cells. Many of the nuclear proteins participating in the life processes tend to concentrate on subnuclear compartments. The subnuclear localization of nuclear proteins is hence important for deeply understanding the construction and functions of the nucleus. Recently, Gene Ontology (GO) annotation has been used for prediction of subnuclear localization. However, the effective use of GO terms in solving sequence-based prediction problems remains challenging, especially when query protein sequences have no accession number or annotated GO term. This study obtains homologies of query proteins with known accession numbers using BLAST to retrieve GO terms for sequence-based subnuclear localization prediction. A prediction method PGAC, which involves mining informative GO terms associated with amino acid composition features, is proposed to design a support vector machine-based classifier. PGAC yields 55 informative GO terms with training and test accuracies of 85.7% and 76.3%, respectively, using a data set SNL_35 (561 proteins in 9 localizations) with 35% sequence identity. Upon comparison with Nuc-PLoc, which combines amphiphilic pseudo amino acid composition of a protein with its position-specific scoring matrix, PGAC using the data set SNL_80 yields a leave-one-out cross-validation accuracy of 81.1%, which is better than that of Nuc-PLoc, 67.4%. Experimental results show that the set of informative GO terms are effective features for protein subnuclear localization. The prediction server based on PGAC has been implemented at http://iclab.life.nctu.edu.tw/prolocgac.     

The primary structure of yeast mitochondrial tyrosine tRNA

The mitochondrial tyrosine tRNA from Saccharomyces cerevisiae has been sequenced. It has two interesting structural features: (i) it lacks two semi-invariant purine residues in the D-loop which are involved in tertiary interactions in the yeast cytoplasmic tRNAPhe; (ii) it has a large variable loop and therefore resembles procaryotic tRNAsTyr rather than eucaryotic cytoplasmic ones.     

Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.