This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Many of the protein-protein interactions involved in transcription have been elucidated from studies on genes transcribed by RNA polymerase II, and serve as paradigms for understanding the various mechanisms involved in the regulation of transcription. However, transcription by RNA polymerase I involves an independently evolved group of transcription factors. Understanding the interactions among these factors may provide new insights into the mechanisms of gene transcription. The long-term goal of our work is to determine the protein-protein interactions that regulate ribosomal RNA (rDNA) transcription. The ability of RNA polymerase I (pol I) itself to participate in rDNA transcription is regulated by the assembly of the core subunits of pol I with different polymerase associated factors. Previous data show at least two polymerase-associated factors are required for transcription. One of these, Rrn3, must be phosphorylated to function in transcription.1,2 We hypothesize that the state of Rrn3 phosphorylation regulates the formation of the Rrn3-RNA polymerase I complex that is required for transcription. The goal of this project is to test this hypothesis by identifying the phosphorylated residues in Rrn3 and determining their role(s) in transcription. Part of this goal includes the characterization of the interactions between Rrn3 and the other components of the rDNA transcription apparatus.Definitive identification of phosphorylated peptides and specific amino acids can be obtained by MALDI-TOF-MS, LC-MS/MS or nanospray-MSn analysis. Peptides generated with trypsin or relatively nonspecific proteases, chymotrypsin and V8 protease, from active Rrn3 purified from exponentially growing cells will be analyzed by mass spectrometry to identify the phosphorylation sites. It may be feasible to facilitate the MS analyses by carrying out 2D-TLC to identify tryptic fragments and then subject just those spots to MS/MS (similar to LC-MS). In preliminary experiments, we have determined that we can purify enough protein by this method to carry out subsequent MS analysis. Furthermore, tryptic peptides from Rrn3 were analyzed at the BUSM MSR by electrospray FTMS using the newly developed ESI-qQq-FTMS. One peptide, tryptic peptide T36-48, was phosphorylated, but the abundance of this peptide in the spectrum was ~3% of the most abundant ion. The region around m/z 404 was isolated using Q1 and accumulated in Q2 prior to electron capture dissociation and collisionally activated dissociation. The combined b/y and c/z cleavages from the two methods multiply confirmed the site of phosphorylation. Overall, the magenerated on the basis of ECD and CAD FTMS analysis is well populated, but further work using IMAC to enrich the phosphopeptides prior to ECD, and using different enzymes such as Asp-N and Glu-C should improve the coverage.The accuracy of mass measurements with delayed extraction and a reflector analyzer (accurate to ~10 parts per million with intense peaks), make it possible to unambiguously assign phosphopeptides of a protein with a known sequence such as Rrn3. Peptides measuring 80 Da higher in mass than their predicted sequence are tentatively marked as phosphopeptides to be confirmed by MS/MS. Confirmation of phosphorylation can also be obtained by comparing the MALDI spectra obtained before and after digestion with alkaline phosphatase. Phosphorylated peptides can also be analyzed using nanoflow-HPLC-MS/MS. When the MS/MS is run in positive ion mode, peptide whole masses (MS) are measured and data for peptide sequencing is obtained by operating a Q-TOF-MS in the data-dependent mode and cycling through MS and MS/MS experiments. In a neutral ion loss experiment a precursor ion scan is used to identify those parent ions that undergo the characteristic 98 Dalton ion neutral loss corresponding to the elimination of phosphoric acid. At this time we have been able to achieve ~58% coverage of Rrn3 isolated from Sf9 cells and have identified four phosphopeptides. Two of these peptides, ALENDFFNSPPR and EGDVDVSDSDDEDDN LPANFDTCHR, are the same as those identified by Schlosser et al. We are now working with Prof. O'Connor to achieve complete PTM analysis of Rrn3 using top-down MS/MS, high resolution bottom-up MS/MS, and electron capture dissociation on his FTMS. While Rrn3 is clearly a bit large for most Top-Down experiments, his new ESIi-qQq-FTMS has the ability to selectively accumulate ions and perform a wide variety of MS/MS methods that may improve and simplify this analysis. Already some signal has been observed and some phosphopeptides from Rrn3 have been observed by ESI-FTMS.A publication is being prepared.
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