bRecent 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.
RNA polymerase II (RNA pol II) produces all mRNAs and many noncoding RNAs but contributes less than 10% of the total RNA present in growing cells (1). It consists of 12 protein subunits with a heterodimeric subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). The catalytic core of the bacterial and eukaryotic enzymes is highly conserved through evolution. However, only five subunits have bacterial homologs (Rpb1, Rpb2, Rpb3, Rpb6, and Rpb11); the others are common to archaea but have no eubacterial homologs (2, 3). The RNA pol II transcription machinery is the most complex of those associated with the three RNA polymerases, with a total of nearly 60 polypeptides, including general transcription factors, coregulators, and specific transcription activators as well as repressors (1).Many studies have contributed to the knowledge of physical interactions between RNA pol II and transcriptional regulators and have enabled the identification of regions that are important for transcription, from initiation to mRNA export (2, 4-12). In addition, we have recently reported the existence of five "conserved domains," located at the surface of the structure of the complex, with poor or no conservation in their paralogs in RNA polymerases I (Rpa190 and Rpa135) and III (Rpc160 and Rpc128) and in their homologs in archaea and bacteria and demonstrate that all of them make contact with transcriptional regulators (10).One of these regions corresponds to the foot domain (2, 10), which, in cooperation with the "lower jaw," the "assembly" domain, and the "cleft" regions, constitutes the "shelf" module of RNA pol II, which might contribute to the rotation of the DNA as it advances toward the active center (2,8). This domain, conserv...