POST-TRANSLATIONAL PROTEIN MODIFICATION AND RNA PROCESSING AND DECAY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Structural studies of mRNA capping & SUMO protein modification. mRNA capping. Three catalytic steps are required in all eukaryotic organisms to properly cap the nascent mRNA chain. The 5' triphosphate (pppN) of the mRNA is cleaved by RNA triphosphatase at the ¿ position to produce a 5' diphosphate (ppN) mRNA molecule. The product of this reaction is substrate for RNA guanylyltransferase in a reaction that transfers GMP from GTP to the 5' diphosphate end of the RNA producing GpppN. The cap guanylate is then methylated by RNA (guanine-7) methyltransferase to form the functional m7GpppN structure. Each of the cap- forming activities is essential for cell growth in budding yeast. We are characterizing the structural basis for several of these enzymes in complex with each other, in complex with various RNA and oligonucleotide compounds, and in complex with the phosphorylated CTD from RNA polymerase II. SUMO. The small ubiquitin-like modifier SUMO is known to regulate nuclear transport, stress response, and signal transduction in eukaryotes, a process that is essential for cell cycle progression in yeast. Analogous to ubiquitin modification, SUMO conjugation occurs on lysine residues and is catalyzed by E1, the SUMO activating enzyme, E2, the SUMO conjugation enzyme, E3-like conjugation cofactors, and proteases that catalyze SUMO processing and deconjugation. SUMO modification does not appear to target proteins for degradation, but rather alters the target protein function through changes in cellular localization, biochemical activation, or through protection from ubiquitin-dependent degradation. We have structurally characterized several components of this system, both alone and in complex with each other. We are currently characterizing the structural basis for additional complexes between E1, E2, E3, SUMO and various substrates and cofactors.