The broad goal of this proposal is to elucidate the structural details of the numerous protein-RNA interactions that are essential in the recognition and modification of eukaryotic messenger RNA. The nascent eukaryotic transcript is subjected to numerous modifications before the journey out of the nucleus. As transcripts are necessarily diverse in sequence, general recognition of the single stranded RNA messages must be sequence non-specific. Modifications at the 5' end of the message aid in transcript recognition by providing unique molecular handles for cellular proteins. In this proposal, we will focus on the recognition of one such molecular handle (the 5' m7G cap), the further modification of this handle (the O2-methylation of the first transcribed nucleotide), and the general sequence non- specific recognition of the transcript itself. These events will be studied in three model systems by combining the structural information of X-ray crystallographic analysis with the functional information obtained from biochemical assays. The concerted application of structural and functional analysis on structurally diverse but functionally similar proteins is a powerful method for elucidating structural features essential to protein function.
Specific aim 1 is to elucidate the structural determinants of m7G cap binding and discrimination using the cap-specific RNA methyltransferase VP39 and the guanosine specific ribonuclease T1 as model systems.
Specific aim 2 is to test the mechanism for single-stranded sequence non-specific RNA binding observed in a recent VP39/RNA co-crystal structure and then to determine the generality of this mechanism through the structural and biochemical characterization of a monoclonal anti-RNA antibody D44.
Specific aim 3 examines the structural details and mechanism of O2 ribose RNA methylation by inducing catalysis in a VP39/RNA co- crystal. The results of these studies will prove invaluable in the analysis of current functional information and future structural information obtained from cellular RNA processing machinery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058692-02
Application #
6181272
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Chin, Jean
Project Start
1999-08-01
Project End
2004-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
2
Fiscal Year
2000
Total Cost
$239,464
Indirect Cost
Name
Emory University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Ratner, Gary A; Hodel, Alec E; Powers, Maureen A (2007) Molecular determinants of binding between Gly-Leu-Phe-Gly nucleoporins and the nuclear pore complex. J Biol Chem 282:33968-76
Hodel, Alec E; Hodel, Mary R; Griffis, Eric R et al. (2002) The three-dimensional structure of the autoproteolytic, nuclear pore-targeting domain of the human nucleoporin Nup98. Mol Cell 10:347-58
Hsu, P C; Hodel, M R; Thomas, J W et al. (2000) Structural requirements for the specific recognition of an m7G mRNA cap. Biochemistry 39:13730-6