The Interaction Between Actin And Rpb5, Rpb6 And Rpb7 RNA Polymerase Ⅱ Subunits

Actin is the component of the cytoskeletal system that allows movement of cells and cellular processes. Actin is an ancient and abundant protein in the typical eukaryotic cell, accounting for about 15% in some cell types, with well established roles in fundamental processes ranging from cell migration to membrane transport. Once thought to be simply a component of muscle, actin is now known to be a ubiquitous constituent of eukaryotic cells. The body of evidence that is summarized above indicates that actin is present in the nucleus in many kinds of eukaryotic cells and implicates it in diverse functions, including transcription, nucleocytoplasmic transport, and chromatin and nuclear structure.Recent studies performed in vitro have suggested that actin, in direct contact with the transcription apparatus, is required in an early step of transcription that is common to all three eukaryotic RNA polymerases. But its function is still not clear and its role has been an issue of interest.The area in which much more needs to be learned is how actin interacts with all three transcription machineries. This includes both the many subunits of all three polymerases and the many transcription factors and other accessory proteins for each polymerase, this may help to define how actin works in each case and, at the most optimistic, such studies may reveal common features that will provide a fundamental insight as to how this ancient protein has collaborated with gene transcription during eukaryotic evolution.In this study we constructed the recombinant pcDNA-HA-rpb in E.coli cells and we expressed the three polymerase II subunits; Rpb5, Rpb6 and Rpb7 as HA-tagged polypeptides by transfection in 293T mammalian cells, as well as untagged nuclearβ-actin. The colocalization ofβ-actin with RNAP II subunits demonstrated with immunofluorescence in HeLa cells. Meanwhile actin can be immunoprecipetated with anti HA-antibody, indicating the interaction in vivo. Glutathione S-transferase (GST) fusion proteins of Rpb5, Rpb6 and Rpb7 were expressed and purified in E.coli and we tested the interaction ofβ-actin with RNAP II subunits in vitro by GST pull-down assay. However, all the three polymerase II subunits tested can interact with actin in vivo. Except for Rpb6; we found that nuclear actin can associate with Rpb5, Rpb7 in vitro. Together, we demonstrated that nuclear actin can associate with individual polymerase II subunits; the results described thus far suggest thatβ-actin is associated very tightly with the polymerse II complex, probably through direct protein-protein interactions with one of more of the Rpb5, Rpb6 and Rpb7 subunits.