Yeast tRNA ligase (Trl1) converts cleaved tRNA halfmolecules into spliced tRNAs containing a 2-PO 4 , 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-PO 4 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-PO 4 , and 5-PO 4 ends are sealed by an ATP-dependent RNA ligase. ) 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).Intron-containing tRNAs are widespread in the archaeal and eukaryal domains of the universal phylogenetic tree (1). The intron is usually located in the anticodon loop of the pre-tRNA and must be removed precisely for the tRNA to function in protein synthesis. tRNA splicing occurs in two stages: (i) intron excision and (ii) joining of the broken tRNA halves (2, 3) (Fig. 1). Unlike pre-mRNA splicing in eukarya, which relies on ribonucleoprotein catalysts, all of the reactions of the tRNA splicing pathway are performed by protein enzymes. The intron removal phase of tRNA splicing requires two incisions of the pre-tRNA at the exon-intron borders. The chemistry of the reaction entails breakage of the phosphodiester backbone by transesterification to yield 2Ј,3Ј-cyclic phosphate and 5Ј-OH termini at both incision sites (2). The breakage reactions are catalyzed by a tRNA splicing endonuclease that specifically recognizes the fold of the pre-tRNA (4 -10). The specificity and fidelity of tRNA splicing are largely governed by the endonuclease component, which is conserved in structure and mechanism among archaea and lower and higher eukaryal species (4 -10).The joining phase of the tRNA splicing pathway has been studied most extensively in yeast, where a single multifunctional tRNA ligase enzyme (Trl1) catalyzes a series of chemical transformations at the ends of the broken tRNA half-molecules that eventuates in the formation of a ligated tRNA molecule containing a 2Ј-PO 4 , 3Ј-5Ј phosphodiester structure at the junction (3, 11). Trl1 performs three reactions: (i) the 2Ј,3Ј-cyclic phosphate terminus is hydrolyzed to a 3Ј-OH, 2Ј-PO 4 terminus by a 2Ј,3Ј-cyclic phosphodiesterase (CPD) 1 activity; (ii) the 5Ј-OH terminus is phosphorylated by an NTP-dependent polynucleotide kinase activity; and (iii) the resulting 3Ј-OH, 2Ј-PO...

Yeast tRNA ligase (Trl1) is an essential enzyme that converts cleaved tRNA half-molecules into spliced tRNAs containing a 2-PO4, 3-5 phosphodiester at the splice junction. Trl1 also catalyzes splicing of HAC1 mRNA during the unfolded protein response. Trl1 performs three reactions: the 2,3-cyclic phosphate of the proximal RNA fragment is hydrolyzed to a 3-OH, 2-PO4 by a cyclic phosphodiesterase; the 5-OH of the distal RNA fragment is phosphorylated by a GTP-dependent polynucleotide kinase; and the 3-OH, 2-PO4, and 5-PO4 ends are then sealed by an ATP-dependent RNA ligase. The removal of the 2-PO4 at the splice junction is catalyzed by the essential enzyme Tpt1, which transfers the RNA 2-PO4 to NAD ؉ to form ADP-ribose 1-2-cyclic phosphate. Here, we show that the bacteriophage T4 enzymes RNA ligase 1 and polynucleotide kinase͞phosphatase can fulfill the tRNA and HAC1 mRNA splicing functions of yeast Trl1 in vivo and bypass the requirement for Tpt1. These results attest to the portability of RNA-repair systems, notwithstanding the significant differences in the specificities, mechanisms, and reaction intermediates of the individual yeast and T4 enzymes responsible for the RNA healing and sealing steps. We surmise that Tpt1 and its unique metabolite ADP-ribose 1-2-cyclic phosphate do not play essential roles in yeast independent of the tRNA-splicing reaction. Our finding that one-sixth of spliced HAC1 mRNAs in yeast cells containing the T4 RNA-repair system suffered deletion of a single nucleotide at the 3 end of the splice-donor site suggests a model whereby the yeast RNA-repair system evolved a requirement for the 2-PO 4 for RNA ligation to suppress inappropriate RNA recombination. RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase 1 (Rnl1) is the founding member of this class of enzymes (1), which includes yeast tRNA ligase and trypanosome RNA-editing ligases (2, 3). The function of Rnl1 in vivo is to repair a break in the anticodon loop of Escherichia coli tRNA Lys triggered by phage activation of a host-encoded anticodon nuclease PrrC (4, 5). Depletion of tRNA Lys by PrrC blocks phage protein synthesis and arrests the infection before it can spread. However, the bacteriophage T4 enzymes polynucleotide kinase͞phosphatase (Pnkp) and Rnl1 repair the broken tRNAs and thereby thwart the host defense mechanism.The enzymatic steps in bacteriophage tRNA restriction͞repair are broadly similar to those of yeast tRNA splicing, the process whereby introns are removed seamlessly from the tRNA anticodon loop (2). The incision steps in both cases result in the formation of 2Ј,3Ј-cyclic phosphate and 5Ј-OH termini. tRNA splicing requires two breaks in the backbone of the pre-tRNA to excise the intron, whereas tRNA restriction involves a single break in the mature tRNA (Fig. 1). The 2Ј,3Ј-cyclic phosphate and 5Ј-OH ends are not substrates for T4 RNA ligase or yeast tRNA ligase, which seal only 3Ј-OH and 5Ј-PO 4 RNA termi...

RNA guanylyltransferase is an essential enzyme that catalyzes the second of three steps in the synthesis of the 5'-cap structure of eukaryotic mRNA. Here we conducted a mutational analysis of the guanylyltransferase domain of the mouse capping enzyme Mce1. We introduced 50 different mutations at 22 individual amino acids and assessed their effects on Mce1 function in vivo in yeast. We identified 16 amino acids as being essential for Mce1 activity (Arg299, Arg315, Asp343, Glu345, Tyr362, Asp363, Arg380, Asp438, Gly439, Lys458, Lys460, Asp468, Arg530, Asp532, Lys533, and Asn537) and clarified structure-activity relationships by testing the effects of conservative substitutions. The new mutational data for Mce1, together with prior mutational studies of Saccharomyces cerevisiae guanylyltransferase and the crystal structures of Chlorella virus and Candida albicans guanylyltransferases, provide a coherent picture of the functional groups that comprise and stabilize the active site. Our results extend and consolidate the hypothesis of a shared structural basis for catalysis by RNA capping enzymes, DNA ligases, and RNA ligases, which comprise a superfamily of covalent nucleotidyl transferases defined by a constellation of conserved motifs. Analysis of the effects of motif VI mutations on Mce1 guanylyltransferase activity in vitro highlights essential roles for Arg530, Asp532, Lys533, and Asn537 in GTP binding and nucleotidyl transfer.

Tpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2-PO 4 from the splice junction to NAD + to form ADP-ribose 1؆-2؆ cyclic phosphate and nicotinamide. To understand the structural requirements for Saccharomyces cerevisiae Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea. We thereby identified four residues-Arg23, His24, Arg71, and Arg138-as essential for Tpt1 function in vivo. Structure-activity relationships at these positions were clarified by introducing conservative substitutions. The activity of the Escherichia coli ortholog KptA in complementing tpt1⌬ was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125. Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30°C. These findings attest to the structural and functional conservation of Tpt1-like 2-phosphotransferases and identify likely constituents of the active site.