The tRNAHis guanylyltransferase (Thg1) is absolutely essential in yeast, and likely throughout all eukaryotes, due to the universal requirement for G-1 on tRNAHis in all eukaryotes in which it has been investigated. Thg1 adds G-1 to tRNAHis via an unusual non-templated 3'-5'nucleotide addition reaction, by an unknown catalytic mechanism that cannot be predicted based on similarity to known enzymes, and thus is likely to employ a novel catalytic mechanism. Moreover, we have recently demonstrated that all Thg1 family members catalyze a template-dependent 3'-5'addition reaction with various substrates, and that this activity is used for a form of G-1 addition in archaea, as well as for an unusual tRNA editing reaction in protozoa. These demonstrated roles for templated 3'-5'addition greatly expand the scope of catalytic activities exhibited by Thg1 family members. Nonetheless, the presence of Thg1 homologs in archaea and bacteria that do not require enzymatic G-1 addition to tRNAHis and unexplained Thg1-related phenotypes in yeast and human cells suggest that additional roles for 3'-5'addition are yet to be uncovered. This application proposes the use of kinetic, genetic, biochemical and structural techniques to investigate the molecular mechanisms and biological functions of both non-templated and templated 3'-5'addition reactions catalyzed by diverse Thg1 family members. These results will provide insight into catalysis of a novel and apparently widespread, but largely unexplored, reaction in biology, and will enable further investigation into alternative functions for 3'-5'nucleotide addition in biological systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087543-05
Application #
8699201
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Barski, Oleg
Project Start
2010-08-15
Project End
2015-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
5
Fiscal Year
2014
Total Cost
$287,952
Indirect Cost
$94,902
Name
Ohio State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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Edvardson, Simon; Elbaz-Alon, Yael; Jalas, Chaim et al. (2016) A mutation in the THG1L gene in a family with cerebellar ataxia and developmental delay. Neurogenetics 17:219-225
Rao, Bhalchandra S; Jackman, Jane E (2015) Life without post-transcriptional addition of G-1: two alternatives for tRNAHis identity in Eukarya. RNA 21:243-53
Jackman, Jane E (2015) An origin story: ribozyme catalysis by the ribosome. RNA 21:650-1
Long, Yicheng; Jackman, Jane E (2015) In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions. FEBS Lett 589:2124-30
Smith, Brian A; Jackman, Jane E (2014) Saccharomyces cerevisiae Thg1 uses 5'-pyrophosphate removal to control addition of nucleotides to tRNA(His.). Biochemistry 53:1380-91
Abad, Maria G; Long, Yicheng; Kinchen, R Dimitri et al. (2014) Mitochondrial tRNA 5'-editing in Dictyostelium discoideum and Polysphondylium pallidum. J Biol Chem 289:15155-65
Betat, Heike; Long, Yicheng; Jackman, Jane E et al. (2014) From end to end: tRNA editing at 5'- and 3'-terminal positions. Int J Mol Sci 15:23975-98
Jackman, Jane E; Alfonzo, Juan D (2013) Transfer RNA modifications: nature's combinatorial chemistry playground. Wiley Interdiscip Rev RNA 4:35-48
Hyde, Samantha J; Rao, Bhalchandra S; Eckenroth, Brian E et al. (2013) Structural studies of a bacterial tRNA(HIS) guanylyltransferase (Thg1)-like protein, with nucleotide in the activation and nucleotidyl transfer sites. PLoS One 8:e67465

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