Pseudouridine (psi), the most common modified nucleoside in RNA, occurs in ribosomal RNA (rRNA) ( transfer RNA (tRNA), and small nuclear (sn) and nucleolar (sno) RNAs, but not in messenger RNA. Psi is made by enzyme-catalyzed isomerization of specific uridines in a pre-formed polynucleotide. This unique reaction involves breaking the uridine N-C glycosyl bond, rotating the uracil base, and reforming a C-C glycosyl link. Two classes of enzyme systems exist. In eubacteria, all psi are made by a set of site-specific proteins. In archaea and eukarya, psi in tRNA and some sn(o) RNAs are made by specific proteins also, but psi in rRNA and other sn(o)RNAs are made by a ribonucleoprotein complex containing a guide RNA for site selection. Genes for psi synthases occur in the most ancient genomes, indicating that ( was an old invention that has persisted for millennia. In this work, the role of psi and psi synthases will be studied using E, coli as the model organism. E. coli has 11 psi sites in rRNA and 7 in tRNA. The psi are made by 11 different synthases because some make multiple psi. The only synthase deletion which markedly inhibited growth was that of RluD, a synthase responsible for 3 psi found at the same place in almost all 50S ribosomes. 50S ribosomes from this strain were highly abnormal, and appeared blocked at some stage of assembly. We plan to (1) study the mechanism by which deletion of RluD blocks ribosome assembly, (2) determine the X-ray structure of RluD-substrate complexes in order to understand the nature of this reaction, (3) study the features of the RNA substrate that allow specific recognition by RluD, and (4) look for physical and/or functional partners of RluD and the other synthases by a synthetic lethal screen and by searching for association in the cell by """"""""pull-down"""""""" experiments. This work will contribute to our understanding of the basic biology of the cell. Moreover, since RluD is so necessary for growth and appears absent in eukaryotes, potential exists for the development of antibacterial therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058879-08
Application #
7070043
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
1999-01-01
Project End
2008-06-30
Budget Start
2006-07-01
Budget End
2008-06-30
Support Year
8
Fiscal Year
2006
Total Cost
$266,292
Indirect Cost
Name
University of Miami School of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
052780918
City
Miami
State
FL
Country
United States
Zip Code
33146
Basturea, Georgeta N; Deutscher, Murray P (2007) Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE. RNA 13:1969-76
Vaidyanathan, Pavanapuresan P; Deutscher, Murray P; Malhotra, Arun (2007) RluD, a highly conserved pseudouridine synthase, modifies 50S subunits more specifically and efficiently than free 23S rRNA. RNA 13:1868-76
Basturea, Georgeta N; Rudd, Kenneth E; Deutscher, Murray P (2006) Identification and characterization of RsmE, the founding member of a new RNA base methyltransferase family. RNA 12:426-34
Del Campo, Mark; Recinos, Claudia; Yanez, Giscard et al. (2005) Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans. RNA 11:210-9
Kaya, Yusuf; Del Campo, Mark; Ofengand, James et al. (2004) Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold. J Biol Chem 279:18107-10
Del Campo, Mark; Ofengand, James; Malhotra, Arun (2004) Crystal structure of the catalytic domain of RluD, the only rRNA pseudouridine synthase required for normal growth of Escherichia coli. RNA 10:231-9
Kaya, Yusuf; Ofengand, James (2003) A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya. RNA 9:711-21
Del Campo, Mark; Ofengand, James; Malhotra, Arun (2003) Purification and crystallization of Escherichia coli pseudouridine synthase RluD. Acta Crystallogr D Biol Crystallogr 59:1871-3
Ofengand, James (2002) Ribosomal RNA pseudouridines and pseudouridine synthases. FEBS Lett 514:17-25
Gutgsell, N S; Del Campo, M D; Raychaudhuri, S et al. (2001) A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain. RNA 7:990-8

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