RNA polymerases (RNAP) play a crucial role in cellular processes by acting as an intermediary between genes and their end products, proteins. Simply put, RNA polymerases transcribe DNA to RNA; RNA is translated into protein. With the crucial role that RNA polymerases play, it is easy to see why they are found in every organism, and even some viruses. The three main eukaryotic RNAPs are RNAP I, II and III. All three are macromolecular complexes consisting of approximately 12 polypeptide subunits. Although RNAP II has been the most studied, all RNAPs interact with other proteins forming temporary complexes with specific functions
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In an article published in the February 2012 (epub December 2011) issue of the Journal of Proteome Research, Daniels, et al. used HaloTag® technology to isolate all three RNAP complexes, capture interacting proteins and characterize the complexes' location. To do this, they created HaloTag® fusion constructs using a subunit common to all three RNAP, POL2RH.
Using the HaloTag®-POL2RH fusion constructs, the authors were able to capture the core subunits for all three RNAPs. In addition, they were able to identify several known associated proteins, which suggests that the HaloTag®-POL2RH fusion protein is incorporated into active complexes in vivo. Perhaps most excitingly, they also identified three new interacting proteins, MLLT11, LOC85395 and LOC90488. These proteins were consistently identified in the protein pull-down pools.
Thanks to the unique multifunctionality of the HaloTag® technology
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, the same fusion protein used to identify the three new interacting proteins also could be used for cellular imaging studies to determine where these proteins are localized. In addition to the HaloTag®-POL2RH fusion construct, fusion constructs also were created for MLLT11, LOC85395 and LOC90488. These constructs were transfected into O2OS cells and then labeled with the HaloTag® TMR Ligand twenty-four hours post transfection. Next, an anti-POLR2A antibody and an Alexa Fluor® 488 fluorescent secondary antibody was applied to show localization with endogenous RNAP II.
The imaging results showed that the HaloTag®-POL2RH fusion protein was localized primarily in the nucleus, matching POLR2A. The three new interacting proteins had much different localization patterns. Both MLLT11 and LOC90488 matched the nuclear localization of POLR2A but also showed localization to the cytoplasm. LOC85395 was restricted to the nucleus, but unlike POLR2A showed localization throughout the nucleus.
The crucial role that RNAPs play in cellular processes makes any new interacting proteins important for further study. Aside from identifying and localizing three new RNAP interacting proteins, the authors also demonstrate the power and versatility of the HaloTag® Technology for isolating and characterizing proteins. The integrated research approach demonstrated by Daniels, et al. shows great promise for accelerating protein research.