Through all kingdoms of life RNAs are modified as, or after, they are synthesized. The types and sites of modification are often conserved, implying conservation of function. Approximately 0.8% of the coding capacity of the E. coli cell is dedicated to encoding enzymes that modify RNA. Many of these modifications are located at key functional regions of the ribosome or other RNAs. The enzymes that carry out the modifications exhibit unique or limited multisite specificity.
The aim of this proposal is to determine the basis for selectivity and catalysis in three families of RNA modifying enzymes: 5-methyl uracil (m5U) methyltransferases (MTases), 5-methyl cystosine (mSC) MTases, and pseudouridine synthases (Psi synthases). These families evoke some of the most common conserved modifications of the more than 80 different types seen in RNA. A common feature of the chemical mechanisms of these families is Michael addition of an amino acid nucleophile to C-6 of the target pyrimidine to activate C-5 for alkylation. Each modification is evoked at a particular stage in RNA folding suggesting that the determinants of selectivity often involve unique three-dimensional folded structures. We seek to determine X-ray crystal structures of several members of each family to elucidate the basis for three-dimensional selectivity in enzyme-RNA targeting. The strategy is to express each enzyme from E. coli and orthologs from two other species, B. subtilis and T. maritima, to provide many candidates for crystallization trials. A minimal, often 17-50 base RNA substrate is sought. A synthetic minimal substrate, containing 5-fluorofoyrimidine at the target site, when reacted with the protein yields a covalent protein-substrate complex, providing an effective means to crystallize enzyme-RNA complexes. The structures of these are compared to structures of enzymes alone or with cofactors. The structures are the basis for the design of mutations in the enzyme or the substrate, and for computational evaluations. Biochemical and structural analysis of the mutant enzymes or substrates further elucidate contributors to the high selectivity and enzyme mechanism. An unrelated RNA MTase, human m1A58 tRNA MTase, will also be subject to structural determination to facilitate structure-based drug discovery. This is a required enzyme in HIV replication, and as such it may represent a new target for anti-HIV drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051232-12
Application #
7220658
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
1996-05-01
Project End
2009-04-30
Budget Start
2007-05-01
Budget End
2009-04-30
Support Year
12
Fiscal Year
2007
Total Cost
$270,061
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Finer-Moore, Janet; Czudnochowski, Nadine; O'Connell 3rd, Joseph D et al. (2015) Crystal Structure of the Human tRNA m(1)A58 Methyltransferase-tRNA(3)(Lys) Complex: Refolding of Substrate tRNA Allows Access to the Methylation Target. J Mol Biol 427:3862-76
Lohse, Matthew B; Rosenberg, Oren S; Cox, Jeffery S et al. (2014) Structure of a new DNA-binding domain which regulates pathogenesis in a wide variety of fungi. Proc Natl Acad Sci U S A 111:10404-10
Czudnochowski, Nadine; Ashley, Gary W; Santi, Daniel V et al. (2014) The mechanism of pseudouridine synthases from a covalent complex with RNA, and alternate specificity for U2605 versus U2604 between close homologs. Nucleic Acids Res 42:2037-48
Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv et al. (2013) Development and binding mode assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-?-glutamyl-D-glutamic acid (BGC 945), a novel thymidylate synthase inhibitor that targets tumor J Med Chem 56:5446-55
Czudnochowski, Nadine; Wang, Amy Liya; Finer-Moore, Janet et al. (2013) In human pseudouridine synthase 1 (hPus1), a C-terminal helical insert blocks tRNA from binding in the same orientation as in the Pus1 bacterial homologue TruA, consistent with their different target selectivities. J Mol Biol 425:3875-87
Wang, Zhen; Sapienza, Paul J; Abeysinghe, Thelma et al. (2013) Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site. J Am Chem Soc 135:7583-92
Wang, Zhen; Abeysinghe, Thelma; Finer-Moore, Janet S et al. (2012) A remote mutation affects the hydride transfer by disrupting concerted protein motions in thymidylate synthase. J Am Chem Soc 134:17722-30
Rosenberg, Oren S; Dovey, Cole; Tempesta, Michael et al. (2011) EspR, a key regulator of Mycobacterium tuberculosis virulence, adopts a unique dimeric structure among helix-turn-helix proteins. Proc Natl Acad Sci U S A 108:13450-5
Alian, Akram; DeGiovanni, Andrew; Griner, Sarah L et al. (2009) Crystal structure of an RluF-RNA complex: a base-pair rearrangement is the key to selectivity of RluF for U2604 of the ribosome. J Mol Biol 388:785-800
Alian, Akram; Lee, Tom T; Griner, Sarah L et al. (2008) Structure of a TrmA-RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases. Proc Natl Acad Sci U S A 105:6876-81

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