Abstract
Recent papers have provided insight into the cytoplasmic assembly of RNA polymerase II (RNA pol II) and its transport to the nucleus. However, little is known about the mechanisms governing its nuclear assembly, stability, degradation, and recycling. We demonstrate that the foot of RNA pol II is crucial for the assembly and stability of the complex, by ensuring the correct association of Rpb1 with Rpb6 and of the dimer Rpb4-Rpb7 (Rpb4/7). Mutations at the foot affect the assembly and stability of the enzyme, a defect that is offset by RPB6 overexpression, in coordination with Rpb1 degradation by an Asr1-independent mechanism. Correct assembly is a prerequisite for the proper maintenance of several transcription steps. In fact, assembly defects alter transcriptional activity and the amount of enzyme associated with the genes, affect C-terminal domain (CTD) phosphorylation, interfere with the mRNA-capping machinery, and possibly increase the amount of stalled RNA pol II. In addition, our data show that TATA-binding protein (TBP) occupancy does not correlate with RNA pol II occupancy or transcriptional activity, suggesting a functional relationship between assembly, Mediator, and preinitiation complex (PIC) stability. Finally, our data help clarify the mechanisms governing the assembly and stability of RNA pol II.
ACKNOWLEDGMENTS
We thank M. Werner, F. Estruch, M. Choder, S. Chávez, D. Bentley, S. Buratowski, and R. Parker for providing antibodies. strains. and constructions. We thank the Centro de Intrumentación Científico-Técnico, CICT, Universidad de Jaén for technical support. We also thank F. Estruch and Olga Calvo for critical reading of the manuscript.
This work was supported by grants from the Spanish Ministry of Science and Innovation and FEDER (BFU2010-21975-C03-02 Spain) and from the Junta de Andalucía (BIO258, PI10-CVI6521, and P08-CVI-03508) to F.N. A.I.G.-G. is a recipient of predoctoral fellowships from MEC.