36. Mammalian Cell Lines Expressing Functional RNA Polymerase If Tagged with the Green Fluorescent Protein
Kimihiko Sugaya, Mare Vigneron1 and Peter R. Cook2 (1Institut de Genetique et de Biologie ]VIo1eculaire et Cellulaire (CNRS/INSERM/ULP), France 2The Sir William Dunn School of Pathology Oxford Univ. UK)
Keywords: complementation, green fluorescent protein, RNA polymerase, mutant, transcrtptton
RNA polymerase II is a multi-subunit enzyme responsible for transcription of most enkaryotic genes. It associates with other complexes to form enormous multifunctional 'holoenzymes' involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-senstttve mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcruptton complexes like the wild-type protein; it both reststs extraction with sarkosyl and is hyperphosphorylated at its C-terminus. Remarkably, subunits bearing such a tag can be incorporated into the acttve enzyme, despite the size and complexity of the polym erizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.
Publications:
1)Sugaya, K., Vigneron, M. and Cook, P. R.: J. Cell. S.i, 113, 2679-2683, 2000.
2)Pombo, A., Jones, E., lborra, F. J., Kimura, H. Sugaya, K., Cook, P. R. and Jackson, D. A. Critical ReviewsTM in Eukaryotic Gene Expression, 10 21-29 2000.