Transcription termination is essential, not only to allow the correct ending of functional transcripts but also to prevent interference between adjacent regions of transcription and between transcription and other cellular events such as replication or telomere maintenance. This is even more critical in a compact genome as that of S. cerevisiae. Termination is also one of the main strategies that the cell employs to control and limit the extent of pervasive/hidden transcription. In yeast, the main share of hidden transcripts is constituted by CUTs (Cryptic Unstable Transcripts) that we have contributed to discover and the transcription of which is terminated by the Nrd1 complex. In this proposal we aim at studying two aspects of transcription termination: i) the study of the mechanism of Nrd1-dependent transcription termination and ii) the analysis of a novel pathway of transcription termination that we have recently discovered. First we will exploit the biochemical tools that we set up as part of a previous ANR project. We have succeeded in obtaining transcription termination in vitro with purified RNAPII and Sen1p, a key component of the Nrd1 complex. We will explore the requirements for termination using essentially biochemical approaches with purified wild type and mutant Nrd1 complex components. In a second task, also derived from a previous ANR project, we will study the mechanism and functional significance of transcription termination induced by DNA-binding proteins, road-block termination. We have already shown that the DNA binding protein Reb1p (and possibly other proteins containing a similar DNA binding domain) can induce transcription termination in vivo, and have described the features that distinguish this pathway from the more “canonical” termination pathways. We will further study the mechanism and explore the genomewide distribution of road-block termination with an approach designed to specifically identify RNAPII pausing sites that are resolved by ubiquitination of the large subunit and proteasome degradation. Finally, we will investigate if road-block termination can be observed with other DNA-binding factors of the same family. We believe that this is an ambitious but realistic project in the light of the many, exciting preliminary results already obtained. These studies are expected to further our understanding of the control of pervasive transcription in yeast and possibly other organisms.