NSF Award
0315684
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.<br/>(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.<br/>(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.<br/>(3) The functional role of TruD and its product, pseudouridine 13 in tRNA, will be addressed using genetic and physiological experiments. <br/><br/>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.
