Correct Assembly of RNA Polymerase II Depends on the Foot Domain and Is Required for Multiple Steps of Transcription in Saccharomyces cerevisiae

Abstract

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...

Subunits Common to RNA Polymerases

“…In addition, the partial dissociation of Rpb4/Rpb7 dimmer leads to an increase in mRNA stability by loss of mRNA imprinting [65,66]. Notably, all these defects are overcome by RPB6 overexpression and agree with previous data pointing to an important role of Rpb6 in RNA pol II integrity/assembly [47,[63][64][65].…”

“…Rpb6 and its bacterial homologue have been proposed to promote RNA pol II assembly and/or increase RNA pol stability, through specific interactions with the RNA pol II largest subunit, Rpb1, in the case of S. cerevisiae [53,56,63,64]. It has been recently reported that mutations in foot conserved domain of Rpb1 cause an integrity defect of the RNA pol II, altering the association between Rpb1 and Rpb6, and the correct association of the dimer Rpb4/7.…”

“…It has been recently reported that mutations in foot conserved domain of Rpb1 cause an integrity defect of the RNA pol II, altering the association between Rpb1 and Rpb6, and the correct association of the dimer Rpb4/7. This assembly alteration causes a transcriptional defect, which affects the amount of enzyme associated with genes and its transcriptional activity [64]. In addition, the partial dissociation of Rpb4/Rpb7 dimmer leads to an increase in mRNA stability by loss of mRNA imprinting [65,66].…”

Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

“…Meanwhile, transcription of the C160-encoding gene is unaffected in this mutant (data not shown), so the largest Pol III subunit is probably degraded when unassembled to the complex. Nuclear degradation of the largest Pol II subunit by an Asr1-independent mechanism was detected in mutants defective in the assembly of the Pol II complex (47). Neither the mechanism of C160 degradation nor the location where this process occurs in rpc128-1007 cells is known, and this requires further study.…”

β-Actin regulates interleukin 6-induced p21 transcription by interacting with the Rpb5 and Rpb7 subunits of RNA polymerase II

“…However, we did not detect obvious binding affinity between β-actin and Rpb6. A previous study showed that the Rpb6 subunit is part of a subcomplex that also contains two other subunits, Rpb7 and Rpb1 (Ishiguro et al 1998;Garrido-Godino et al 2013). We thus suspect that our results may stem from the fact that Rpb6 is located between Rpb7 and Rpb1, which prevents its interaction with other proteins.…”