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Analysis of Transcriptional Regulatory Complexes

RNA polymerase I, II, III After affinity purification to purify protein complexes from cells, we use the MudPIT and the NSAF, and now dNSAF approach to quantitatively analyze multiprotein transcriptional regulatory complexes. The combination of these approaches provides not only a list of the proteins present but also the abundance of proteins present. This information can be used in a variety of ways. First, a quantitative proteomic analysis of a protein complex can be carried out to gain insight into the forms of the protein complexes and whether or not there are any abundant and poorly characterized new protein protein interactions.

Examples of our use of this approach include a quantitative proteomic analysis of HeLa cell Mediator where four biological replicates of four different Mediator subunits were used to affinity purify the complex (Paoletti et al., 2006). The technology described in this published work is an important foundation for our research where biological replicates of transcriptional regulatory complexes and subassemblies are analyzed in a bait dependent manner and the statistically significant differences in the complexes and subassemblies are determined.

We have begun to apply this approach to RNA polymerase I, II, and III. During the course of this work, we determined the function of a poorly characterized but highly conserved protein, Rtr1, which was interacting with RNA polymerase II (Mosley, Pattenden, et al., 2009). We then pursued the function of Rtr1 in the transcription cycle and found that Rtr1 localized in coding regions between the peaks of Serine5-phosphorylation and Serine2-phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II, Rpb1. Deletion of Rtr1 resulted in an accumulation of the Serine5-phosphorylated form of Rpb1, a decrease in RNA polymerase II transcription, and termination defects. We further demonstrated that Rtr1 is a Serine 5 phosphatase of Rpb1 C-terminal domain (Mosley, Pattenden, et al., 2009). We have also analyzed yeast RNA polymerase I, II, and III using the quantitative proteomics approaches developed in the lab. We demonstrated that we can properly cluster the proteins in theses complexes, determine the relative abundance of proteins in the complexes (Figure 2) and we are able to capture dynamic interactors of the complexes (Mosley et al., 2011)

Banks CA, Lee ZT, Boanca G, Lakshminarasimhan M, Groppe BD, Wen Z, Hattem GL, Seidel CW, Florens L, Washburn MP. (2014) Controlling for gene expression changes in transcription factor protein networks. Mol Cell Proteomics. Jun;13(6):1510-22. doi: 10.1074/mcp.M113.033902. Epub 2014 Apr 10. Abstract

Mosley AL, Hunter GO, Sardiu ME, Smolle M, Workman JL, Florens L, Washburn MP. (2013) Quantitative Proteomics Demonstrates that the RNA Polymerase II Subunits Rpb4 and Rpb7 Dissociate During Transcription Elongation. Mol Cell Proteomics, Jun;12(6):1530-8. Epub 2013 Feb 15 Abstract

Gilmore JM, Sardiu ME, Venkatesh S, Stutzman B, Peak A, Seidel CW, Workman JL, Florens L, Washburn MP. (2012) Characterization of a highly conserved histone related protein, Ydl156w, and its functional associations using quantitative proteomic analyses. Mol Cell Proteomics, Apr;11(4):M111.011544. Epub 2011 Dec 22 Abstract

Daniels DL, Méndez J, Mosley AL, Ramisetty SR, Murphy N, Benink H, Wood KV, Urh M, Washburn MP. (2012) Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics. J Proteome Res, Feb 3;11(2):564-75. Epub 2012 Jan 3 Abstract

Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu D, Conaway RC, Conaway JW, Florens L, Washburn MP. (2006)Quantitative Proteomic Analysis of Distinct Mammalian Mediator Complexes using Normalized Spectral Abundance Factors. Proc. Natl. Acad. Sci. USA, 103(50):18928-33 Abstract

Mosley AL, Pattenden SG, Carey M, Venkatesh S, Gilmore JM, Florens L, Workman JL, Washburn MP. (2009) Rtr1 Regulates the Transition from Serine 5 to Serine 2 Phosphorylation on the RNA Polymerase II C-terminal Domain during Transcription Elongation. Mol. Cell, 34(2):168-78. Abstract

Mosley AL, Sardiu ME, Pattenden SG, Workman JL, Florens L, Washburn MP. (2011) Highly reproducible label free quantitative proteomic analysis of RNA polymerase complexes. Mol. Cell. Proteomics, 10(2):M110.000687. Abstract