Transcription is the first step of gene expression. As such, it is essential that organisms tightly regulate this process to ensure that the genetic programs are performed harmoniously and allow the proper synthesis of the components that the cell requires at a given time and in a given developmental situation. In eukaryotes, three RNA polymerases (Pol) transcribe the genome. Pol II is responsible for the transcription of messenger RNAs and many small non-coding RNAs. As such, it has to respond to the large number of situations encountered by the cell and to adapt the level of transcription of tens of thousands of genes. In eukaryotes, hundreds to a couple of thousands of transcription activators, recognizing sequences located in enhancers or upstream activating sequences, trigger the transcription of the adjacent genes. A large protein complex, the Mediator of transcription, also called Mediator for short, is responsible for the recruitment of Pol II in response to the transcription factors and, together with the general transcription factors, for the assembly of preinitiation complex and ultimately transcription initiation. Mediator is constituted of at least 25 subunits that are engaged in numerous contacts within the complex. Mediator also interacts with partners belonging to other components of the transcription machinery, including general transcription factors and co-activators that modify the chromatin, and with other complexes involved in various nuclear processes. Mediator can thus be compared to an antenna, capable of receiving the activation signals through protein-protein interactions with the hundreds of transcription activators of the eukaryotic cell, integrate these signals and activate transcription by triggering the recruitment of Pol II and possibly some of the general transcription factors. While Mediator has been studied intensively, its complexity has precluded a detailed understanding of how it responds to activators in connection with co-activators, of what is its role in preinitiation complex formation and of how it ultimately recruits Pol II in vivo. The general objectives of this proposal are, using Saccharomyces cerevisiae as a model system, (i) to investigate in vivo how the protein-protein interactions within Mediator transmit the activation signals from activators to the GTFs and Pol II; (ii) to identify the subunits of Mediator that directly contact Pol II and investigate the role of these contacts and; (iii) to understand how Mediator and co-activators cooperate to activate transcription and stimulate preinitiation complex formation. The rationale of the project will be to identify which subunits of Mediator interact with Pol II using a new in vivo cross-linking methodology. We will then produce collections of temperature sensitive mutants in essential subunits of Mediator, targeting in particular those that interact with Pol II. The effect of the mutations on the interactions of the subunits with all their known partners will be tested. In parallel, we will look at the effect of the mutations on the expression of specific genes using RT-qPCR or genome-wide using DNA micro-arrays. We expect that some of the mutations will lead to genome-wide defects while other will impair transcription of subsets of the genome only. Incidentally, microrarrays will allow the identification of the genes that are the most strongly affected, facilitating the characterization of the impaired mechanism. We will also investigate the effect of the Mediator mutations on the association of Mediator itself, the co-activators, the general transcription factors and Pol II on specific genes (in particular those of the methionine regulon) or, when needed, genome-wide. In this project, we would like to concentrate at first on five essential Mediator subunits: Med4, Med7, Med8, Med10 and Med21. Med7 and Med8 were chosen because our preliminary cross-linking results suggest that they interact directly with Pol II and could thus play a major role in its recruitment. In summary, this project aims at gaining an integrated view of the mechanisms of transcription activation and at understanding how Mediator performs this central function in gene expression in vivo. We will look at this process from the initial step of the recruitment of Mediator by activators to Pol II recruitment taking advantage of the huge power of yeast genetics, molecular biology and functional genomics. We anticipate that our work might have implications for the understanding of human diseases that are the consequence of altered regulation of gene expression.