Pseudouridine is the most common post-transcriptional modification of RNA, and arises from the isomerization of uridine catalyzed by the pseudouridine synthases. These enzymes fall into four families that share no global sequence similarity but appear homologous on the basis of their tertiary structures. Several pseudouridine synthases are physiologically critical in prokaryotes and eukaryotes, including the human enzyme dyskerin, the absence of which causes the X-linked disease dyskeratosis congenita. Pseudouridine residues in small nuclear RNA are required for pre-mRNA splicing, an essential function in eukaryotes. Since the families of pseudouridine synthases are so highly divergent, it remains possible that they proceed by different mechanisms, and such differences may eventually be exploited in new classes of antibiotics or provide a better understanding of the disease dyskeratosis congenita. The proposed experiments will elucidate the chemical mechanism followed by two E. coli pseudouridine synthases of different families, RluA and TruB, which are particularly tractable targets for mechanistic work because they handle small RNA substrates (stem-loop oligonucleotides). RNA containing 5-fluorouridine has been used as an inhibitor and mechanistic probe of the pseudouridine synthases, favoring a mechanism involving a Michael addition. Recent results, however, indicate that TruB is not inhibited by 5-fluorouridine in RNA and that an assumed hydrolysis of a proposed intermediate does not occur, leading to doubt concerning the mechanistic conclusions based on earlier studies with the same compound. The proposed experiments will resolve issue concerning 5-fluorouridine in RNA and allow further mechanistic insight. Such insight will also be gained using RNA containing uridine with a 2'-fluoro group, similar to the series of remarkably informative experiments using fluorosugars to probe the mechanism of glycosidases. A combination of NMR, mass spectrometry, site-directed mutagenesis and kinetic analysis supplemented by X-ray crystallography will be employed to examine the effects of the two types of fluorinated RNA as well as site-directed mutagenesis of amino acid residues that appear to play a role in catalysis. Finally, synthesized small RNA substrates will be used to examine the substrate specificity of RluA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059636-06
Application #
6986788
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
1999-08-01
Project End
2008-11-30
Budget Start
2005-12-31
Budget End
2006-11-30
Support Year
6
Fiscal Year
2006
Total Cost
$228,550
Indirect Cost
Name
University of Delaware
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
059007500
City
Newark
State
DE
Country
United States
Zip Code
19716
Veerareddygari, Govardhan Reddy; Singh, Sanjay K; Mueller, Eugene G (2016) The Pseudouridine Synthases Proceed through a Glycal Intermediate. J Am Chem Soc 138:7852-5
Miracco, Edward J; Bogdanov, Bogdan; Mueller, Eugene G (2011) Unexpected linear ion trap collision-induced dissociation and Fourier transform ion cyclotron resonance infrared multi-photon dissociation fragmentation of a hydrated C-glycoside of 5-fluorouridine formed by the action of the pseudouridine synthases RluA Rapid Commun Mass Spectrom 25:2627-32
Miracco, Edward J; Mueller, Eugene G (2011) The products of 5-fluorouridine by the action of the pseudouridine synthase TruB disfavor one mechanism and suggest another. J Am Chem Soc 133:11826-9
McDonald, Marguerite K; Miracco, Edward J; Chen, Junjun et al. (2011) The handling of the mechanistic probe 5-fluorouridine by the pseudouridine synthase TruA and its consistency with the handling of the same probe by the pseudouridine synthases TruB and RluA. Biochemistry 50:426-36
Hamilton, Christopher S; Greco, Todd M; Vizthum, Caroline A et al. (2006) Mechanistic investigations of the pseudouridine synthase RluA using RNA containing 5-fluorouridine. Biochemistry 45:12029-38
Wright, Chapman M; Christman, Glenn D; Snellinger, Ann M et al. (2006) Direct evidence for enzyme persulfide and disulfide intermediates during 4-thiouridine biosynthesis. Chem Commun (Camb) :3104-6
Hoang, Charmaine; Chen, Junjun; Vizthum, Caroline A et al. (2006) Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure. Mol Cell 24:535-45
Mueller, Eugene G (2006) Trafficking in persulfides: delivering sulfur in biosynthetic pathways. Nat Chem Biol 2:185-94
Hoang, Charmaine; Hamilton, Christopher S; Mueller, Eugene G et al. (2005) Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain. Protein Sci 14:2201-6
Hamilton, Christopher S; Spedaliere, Christopher J; Ginter, Joy M et al. (2005) The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB. Arch Biochem Biophys 433:322-34

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