Michel Werner

The Ccr4-Not Complex Independently Controls both Msn2-Dependent Transcriptional Activation—via a Newly Identified Glc7/Bud14 Type I Protein Phosphatase Module—and TFIID Promoter Distribution

ABSTRACT The Ccr4-Not complex is a conserved global regulator of gene expression, which serves as a regulatory platform that senses and/or transmits nutrient and stress signals to various downstream effectors. Presumed effectors of this complex in yeast are TFIID, a general transcription factor that associates with the core promoter, and Msn2, a key transcription factor that regulates expression of stress-responsive element (STRE)-controlled genes. Here we show that the constitutively high level of STRE-driven expression in ccr4-not mutants results from two independent effects. Accordingly, loss of Ccr4-Not function causes a dramatic Msn2-independent redistribution of TFIID on promoters with a particular bias for STRE-controlled over ribosomal protein gene promoters. In parallel, loss of Ccr4-Not complex function results in an alteration of the posttranslational modification status of Msn2, which depends on the type 1 protein phosphatase Glc7 and its newly identified subunit Bud14. Tests of epistasis as well as transcriptional analyses of Bud14-dependent transcription support a model in which the Ccr4-Not complex prevents activation of Msn2 via inhibition of the Bud14/Glc7 module in exponentially growing cells. Thus, increased activity of STRE genes in ccr4-not mutants may result from both altered general distribution of TFIID and unscheduled activation of Msn2.

An Rpb4/Rpb7-Like Complex in Yeast RNA Polymerase III Contains the Orthologue of Mammalian CGRP-RCP

ABSTRACT The essential C17 subunit of yeast RNA polymerase (Pol) III interacts with Brf1, a component of TFIIIB, suggesting a role for C17 in the initiation step of transcription. The protein sequence of C17 (encoded by RPC17) is conserved from yeasts to humans. However, mammalian homologues of C17 (named CGRP-RCP) are known to be involved in a signal transduction pathway related to G protein-coupled receptors, not in transcription. In the present work, we first establish that human CGRP-RCP is the genuine orthologue of C17. CGRP-RCP was found to functionally replace C17 in Δrpc17 yeast cells; the purified mutant Pol III contained CGRP-RCP and had a decreased specific activity but initiated faithfully. Furthermore, CGRP-RCP was identified by mass spectrometry in a highly purified human Pol III preparation. These results suggest that CGRP-RCP has a dual function in mammals. Next, we demonstrate by genetic and biochemical approaches that C17 forms with C25 (encoded by RPC25) a heterodimer akin to Rpb4/Rpb7 in Pol II. C17 and C25 were found to interact genetically in suppression screens and physically in coimmunopurification and two-hybrid experiments. Sequence analysis and molecular modeling indicated that the C17/C25 heterodimer likely adopts a structure similar to that of the archaeal RpoE/RpoF counterpart of the Rpb4/Rpb7 complex. These RNA polymerase subunits appear to have evolved to meet the distinct requirements of the multiple forms of RNA polymerases.

RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

Translational Control by Arginine of Yeast Gene CPA1

Carbamoylphosphate is a common intermediate of the arginine and pyrimidine biosynthetic pathways. In Saccharomyces cerevisiae, two independently regulated CPSases produce carbamoylphosphate for the two biosyntheses (Lacroute et al., 1965). One synthetase CPSase P, is repressed and feedback inhibited by the pyrimidines. This nuclear enzyme, encoded by the URA2 gene, is part of a multienzymatic protein comprising also the aspartate transcarbamylase activity which catalyses the second step of the pyrimidine biosynthesis. The other synthetase, CPSase A is cytosolic and constituted of two nonidentical subunits encoded by the unlinked genes CPA1 and CPA2. The small subunit, the product of gene CPA1, binds glutamine, the physiological nitrogen donor of the enzyme, and transfers its amide nitrogen group to the larger subunit, encoded by CPA2, that catalyses the synthesis of carbamoylphosphate from ammonia (Pierard and Schroter, 1978). Both CPA1 and CPA2 genes are subject to the general control of amino acid biosynthesis. In contrast, the specific repression by arginine acts only upon the expression of gene CPA1. This difference in the regulation of the two subunits leads, under condition of repression by arginine, to a situation where the large subunit is over-produced by a five fold excess over the small one (Pierard et al., 1979).

2 Yeast RNA Polymerase Subunits and Genes

OVERVIEW Yeast RNA polymerases A(I), B(II), and C(III) are organized around a common core of subunits related to the bacterial core enzyme (β′ βα 2 ) and share a set of five small subunits (ABC27, ABC23, ABC14.5, ABC10α, and ABC10β). All these subunits are essential for growth. In addition, each enzyme contains a variable number of enzyme-specific subunits, some of which are not strictly required for growth. Most subunit genes have been cloned, sequenced, and mutagenized to produce null alleles and, for several of them, conditional mutants. A functional map of RNA polymerase active site, taken in a broad sense, has begun to emerge from a combined genetic and biochemical analysis of the large subunits. GENERAL PROPERTIES OF YEAST NUCLEAR RNA POLYMERASES The budding yeast Saccharomyces cerevisiae , with its biochemically and genetically well-characterized transcription apparatus, is currently the most suitable experimental model for a comprehensive study of eukaryotic RNA polymerases (see Sentenac 1985; Gabrielsen and Sentenac 1991 and references therein; Young 1991; Thuriaux and Sentenac 1992). Yeast RNA polymerases are typically eukaryotic in their subunit complexity, and there is a fair degree of functional equivalence between the transcription machinery of yeasts and of metazoan eukaryotes (see, e.g., Struhl 1989; Sawadogo and Sentenac 1990; and other chapters in this volume). We assume, therefore, that what is learned of the yeast enzymes may largely be extrapolated to other eukaryotes. Several chapters of this volume cover important structural and functional features of the RNA polymerases of higher eukaryotes and of associated general transcription factors (Corden...