Biochemical and Structures Studies of tRNA Modificaton and Repair Enzymes

Autor/in	Zhou, Chun
Titel	Biochemical and Structures Studies of tRNA Modificaton and Repair Enzymes
Quelle	(2009), (114 Seiten) PDF als Volltext Verfügbarkeit Ph.D. Dissertation, University of Illinois at Urbana-Champaign
Sprache	englisch
Dokumenttyp	gedruckt; online; Monographie
ISBN	978-1-1095-8171-3
Schlagwörter	Hochschulschrift; Dissertation; Maintenance; Genetics; Microbiology; Biochemistry; Science Instruction; Physiology; Molecular Structure; Comparative Analysis + Suchen Sie Ihr Suchwort? Thesis; Dissertations; Academic thesis; Humangenetik; Mikrobiologie; Biochemie; Teaching of science; Science education; Natural sciences Lessons; Naturwissenschaftlicher Unterricht; Physiologie
Abstract	RNA hypermodifications near the anticodon of tRNA are fundamental for the efficiency and fidelity of protein synthesis. Dimethylallyltransferase (DMATase) catalyzes transfer of a dimethylallyl moiety from dimethylallyl pyrophosphate to N6 of A37 in certain tRNAs. We first determined the crystal structures of "Pseudomonas aeruginosa" DMATase. Surprisingly, the enzyme possesses a central channel spanning the entire width of the enzyme. Later we determined the crystal structures of "Saccharomyces cerevisiae" DMATase-tRNA[superscript Cys] complex in four distinct forms, which provide snapshots of the RNA modification reaction catalyzed by DMATase. The structures reveal that the enzyme recognizes the tRNA substrate through indirect sequence readout. The targeted nucleotide A37 flips out from the anticodon loop of tRNA and flips into the channel in DMATase, where it meets its reaction partner dimethylallyl pyrophosphate, which enters the channel from the opposite end. Structural changes accompanying the transfer reaction taking place in the crystal result in disengagement of DMATase-tRNA interaction near the reaction center. In addition, structural comparison of DMATase in the complex with unliganded bacterial DMATase provides a molecular basis of ordered substrate binding by DMATase. Plant siRNAs and miRNAs are methylated at their 3' ends at the 2' OH position by Hen1, an S-Adomet dependent methyltransferase. Hen1 homologs in animals are shown to be responsible for the methylation of piRNAs, some siRNAs as well as endogenous siRNAs. Interestingly, a subset of bacteria also has a Hen1 homolog. Since bacteria do not have canonical RNAi, Hen1 in bacteria (bHen1) might play other biological roles, Our goals are to find out the biological function of bHen1. We observed that immediately following bHen1, there was a second highly conserved gene ("Pnkp") in the operon. Pnkp has previously been shown to have kinase, phosphatase and adenylyltransferase activity. Therefore Pnkp/Hen1 might be involved in RNA repair. I cloned and expressed both proteins with a pETDuet vector. Pnkp and bHen1 form a heterotetramer in gel filtration column. Purfied Pnkp/bHEN1 efficiently repaired full length tRNAs cleaved by bacterial toxins (colicin E5 and D). Before the broken RNAs were ligated, a methyl group was added to the 2' OH group that participated in the original RNA cut. Due to this methylation, RNAs repaired by bacterial Pnkp/Hen1 could not be cleaved again by the ribotoxins. Thus, unlike eukaryotic Hen1 involved in RNAi, bacterial Hen1 is part of a RNA repair and modification system. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.] (As Provided).
Anmerkungen	ProQuest LLC. 789 East Eisenhower Parkway, P.O. Box 1346, Ann Arbor, MI 48106. Tel: 800-521-0600; Web site: http://www.proquest.com/en-US/products/dissertations/individuals.shtml
Erfasst von	ERIC (Education Resources Information Center), Washington, DC
Update	2017/4/10