title: Kinetic analysis of 3'-5' nucleotide addition catalyzed by eukaryotic tRNA(His) guanylyltransferase.
abstract: The tRNA(His) guanylyltransferase (Thg1) catalyzes the incorporation of a single guanosine residue at the -1 position (G(-1)) of tRNA(His), using an unusual 3'-5' nucleotidyl transfer reaction. Thg1 and Thg1 orthologs known as Thg1-like proteins (TLPs), which catalyze tRNA repair and editing, are the only known enzymes that add nucleotides in the 3'-5' direction. Thg1 enzymes share no identifiable sequence similarity with any other known enzyme family that could be used to suggest the mechanism for catalysis of the unusual 3'-5' addition reaction. The high-resolution crystal structure of human Thg1 revealed remarkable structural similarity between canonical DNA/RNA polymerases and eukaryotic Thg1; nevertheless, questions regarding the molecular mechanism of 3'-5' nucleotide addition remain. Here, we use transient kinetics to measure the pseudo-first-order forward rate constants for the three steps of the G(-1) addition reaction catalyzed by yeast Thg1: adenylylation of the 5' end of the tRNA (k(aden)), nucleotidyl transfer (k(ntrans)), and removal of pyrophosphate from the G(-1)-containing tRNA (k(ppase)). This kinetic framework, in conjunction with the crystal structure of nucleotide-bound Thg1, suggests a likely role for two-metal ion chemistry in all three chemical steps of the G(-1) addition reaction. Furthermore, we have identified additional residues (K44 and N161) involved in adenylylation and three positively charged residues (R27, K96, and R133) that participate primarily in the nucleotidyl transfer step of the reaction. These data provide a foundation for understanding the mechanism of 3'-5' nucleotide addition in tRNA(His) maturation.
authors.affiliation: Department of Biochemistry, Center for RNA Biology and Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, United States.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Smith
authors.forename: Jane E
authors.initials: JE
authors.lastname: Jackman
mesh_terms: D002384:Catalysis; D005057:Eukaryotic Cells / Q000201:enzymology*; D006151:Guanosine / Q000737:chemistry*; D018398:Homeodomain Proteins / Q000737:chemistry; D006801:Humans; D009713:Nucleotidyltransferases / Q000737:chemistry*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry
chemical_list: D018398:Homeodomain Proteins; C471968:RHOXF2 protein, human; D012346:RNA, Transfer, Amino Acyl; D029701:Saccharomyces cerevisiae Proteins; D006151:Guanosine; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C504741:Thg1 protein, S cerevisiae; C065307:tRNA guanylyltransferase; C023198:tRNA nucleotidyltransferase