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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