The Ccr4-not complex regulates Skn7 through Srb10 kinase.

The Ccr4-Not complex is a multifunctional regulatory platform composed of nine subunits that controls diverse cellular events including mRNA degradation, protein ubiquitination, and transcription. In this study, we identified the yeast Saccharomyces cerevisiae osmotic and oxidative stress transcription factor Skn7 as a new target for regulation by the Ccr4-Not complex. Skn7 interacts with Not1 in a two-hybrid assay and coimmunoprecipitates with Not5 in a Not4-dependent manner. Skn7-dependent expression of OCH1 and Skn7 binding to the OCH1 promoter are increased in not4Delta or not5Delta mutants. Skn7 purified from wild-type cells but not from not4Delta cells is associated with the Srb10 kinase. This kinase plays a central role in the regulation of Skn7 by Not4, since increased OCH1 expression in not4Delta cells requires Srb10. These results reveal a critical role for the Ccr4-Not complex in the mechanism of activation of Skn7 that is dependent upon the Srb10 kinase.

A SAGA-independent function of SPT3 mediates transcriptional deregulation in a mutant of the Ccr4-not complex in Saccharomyces cerevisiae.

The conserved multi-subunit Ccr4-Not complex regulates gene expression in diverse ways. In this work, we characterize the suppression of temperature sensitivity associated with a mutation in the gene encoding the scaffold subunit of the Ccr4-Not complex, NOT1, by the deletion of SPT3. We determine that the deletion of SPT3, but not the deletion of genes encoding other subunits of the SAGA complex, globally suppresses transcriptional defects of not1-2. We find that transcriptional activation in not1-2 is associated with increased binding of TFIID and SAGA at promoters of upregulated genes, and this is suppressed by the deletion of SPT3. Interestingly, Spt3p-dependent activation of transcription occurs in not1-2 even if the SAGA complex is disrupted by the deletion of SPT7 that encodes a subunit of SAGA required for its integrity. Consistent with a SAGA-independent function of Spt3p, the deletion of SPT3 displays synthetic phenotypes when combined with a deletion of SPT7. Taken together, our results provide a new view of the Spt3 protein by identifying a SAGA-independent function of this protein that is functionally linked to the Ccr4-Not complex.

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription.

We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10(3) over the error rate determined solely by selectivity (1.8 x 10(-4)). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.

The yeast Ccr4-Not complex controls ubiquitination of the nascent-associated polypeptide (NAC-EGD) complex.

In this work, we determine that the Saccharomyces cerevisiae Ccr4-Not complex controls ubiquitination of the conserved ribosome-associated heterodimeric EGD (enhancer of Gal4p DNA binding) complex, which consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. We show that the EGD complex subunits are ubiquitinated proteins, whose ubiquitination status is regulated during cell growth. Egd2p has a UBA domain that is not essential for interaction with Egd1p but is required for stability of Egd2p and Egd1p. Ubiquitination of Egd1p requires Not4p. Ubiquitination of Egd2p also requires Not4p, an intact Not4p RING finger domain, and all other subunits of the Ccr4-Not complex tested. In the absence of Not4p, Egd2p mislocalizes to punctuate structures. Finally, the EGD complex can be ubiquitinated in vitro by Not4p and Ubc4p, one of the E2 enzymes with which Not4p can interact. Taken together our results reveal that the EGD ribosome-associated complex is ubiquitinated in a regulated manner, and they show a new role for the Ccr4-Not complex in this ubiquitination.

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation.

While initiation of transcription by RNA polymerase III (Pol III) has been thoroughly investigated, molecular mechanisms driving transcription termination remain poorly understood. Here we describe how the characterization of the in vitro transcriptional properties of a Pol III variant (Pol IIIdelta), lacking the C11, C37, and C53 subunits, revealed crucial information about the mechanisms of Pol III termination and reinitiation. The specific requirement for the C37-C53 complex in terminator recognition was determined. This complex was demonstrated to slow down elongation by the enzyme, adding to the evidence implicating the elongation rate as a critical determinant of correct terminator recognition. In addition, the presence of the C37-C53 complex required the simultaneous addition of C11 to Pol IIIdelta for the enzyme to reinitiate after the first round of transcription, thus uncovering a role for polymerase subunits in the facilitated recycling process. Interestingly, we demonstrated that the role of C11 in recycling was independent of its role in RNA cleavage. The data presented allowed us to propose a model of Pol III termination and its links to reinitiation.

Rpa12p, a conserved RNA polymerase I subunit with two functional domains.

Rpa12p is a subunit of RNA polymerase I formed of two zinc-binding domains. The N-terminal zinc region (positions 1-60) is poorly conserved from yeast to man. The C-terminal domain contains an invariant Q.RSADE.T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N-terminal domain fail to grow at 34 degrees C, are sensitive to nucleotide-depleting drugs and become lethal in rpa14-Delta mutants lacking the non-essential RNA polymerase I subunit Rpa14p. They also strongly alter the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific two-hybrid interaction with the second largest subunit. In all these respects, N-terminal deletions behave like full deletions. In contrast, C-terminal deletions retaining only the first N-terminal 60 amino acids are indistinguishable from wild type. Thus, the N-terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I and is the only critical part of that subunit in vivo.