1999 — 2002 |
Ofengand, James |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pseudouridine and Ribosomes @ University of Miami School of Medicine
In all cells, the process of protein synthesis proceeds on ribosomes, one of the most ancient of all macromolecular structures found in the cell. The functional heart of the ribosome is its RNA, one high molecular weight molecule for each subunit, complexed with a characteristic set of proteins. Ribosomal RNA contains a number of modified nucleosides with the isomer uridine called pseudouridine ( psi) being the most prevalent single modification found. Psi residues are not distributed at random but are found at specific sites, many of which recur from species to species. In prokaryotes, specific enzymes are responsible for the placement of psi at particular sites in ribosomal RNA, while in eukaryotes, an elaborate system of guide RNAs has evolved for placing the psi residues correctly. Despite this careful placement of psi at specific sites in the ribosome, the role of psi in ribosome structure nad/or function is unknown. The specific objective of this proposal is an understanding of how psi residues are made in the ribosome and what their role is in its overall workings, using Escherichia coli as the model system. The specific aims are: (1) to determine the role played by psi at particular sites in ribosomal RNA by deleting them one by one and analyzing the effect on the cell's ability to make protein. Specific deletion will be accomplished by disruption of the gene for the specific enzymes responsible for the synthesis. (2) to understand the RNA-protein recognition mechanisms which these enzymes use to pick out one U out of over 500 others. (3) to determine the stage of ribosome formation at which the psi residues are made. (4) to search for possible progenitors of the eukaryotic guide RNAs in E. coli postulating that before they acquired their site selection function they may have functioned as reaction rate enhancers. The long-term goal is to elucidate the mechanisms of ribosomal RNA function in ribosome at the most basic molecular level. As the process of protein synthesis is so fundamental to all cells, information of this kind cannot help but advance our knowledge of the physiology of the cell both in healthy and diseased states.
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2003 — 2007 |
Malhotra, Arun [⬀] Ofengand, James |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
A New Class of Pseudouridine Synthases @ University of Miami School of Medicine
Pseudouridine is the most common modified nucleoside in RNA, and evidence for pseudouridine synthases, the enzymes that make pseudouridine from uridine, have been found in the most ancient of organisms. Currently, no organism is known without putative pseudouridine synthases. Up to now, all pseudouridine synthases could be grouped into four families, three of which are closely related by amino acid sequence homology, and one which is more loosely related. The principal investigator of this project recently discovered a novel class of pseudouridine synthases with no amino acid sequence homology to the previously known ones. There are so far 59 members of this class distributed among all phyla including ancient organisms. Only one of these 59 proteins had a previously known function. The E. coli representative (TruD) of this new class will be studied by the following three approaches. (1) Since the amino acid sequence of TruD bears no relationship to known pseudouridine synthases, three of which have had their crystal structures determined, determination of the structure of TruD and a TruD-substrate complex by X-ray crystallography will be done in order to directly determine how the enzyme folds and how the active site is constructed using an alternative set of amino acids from that in conventional pseudouridine synthases. (2) Determination of the sequence and secondary structure requirements of the tRNA or tRNA fragment substrate for TruD in solution will be done by making mutant versions of the substrate and analyzing for pseudouridine formation and for complex formation with the synthase. These results will complement the static structural information to be obtained from the X-ray structure of TruD-substrate complexes. (3) The functional role of TruD and its product, pseudouridine 13 in tRNA, will be addressed using genetic and physiological experiments.
Broader Impact: The project provides an outstanding opportunity for training graduate students in multidisciplinary research involving genetics, biochemistry and crystallography. The project will use this opportunity creatively in research training at different levels.
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2003 |
Ofengand, James |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pseudouridines, Pseudouridine Synthases, and Ribosomes @ University of Miami School of Medicine
DESCRIPTION (provided by applicant): 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.
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