Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome displacement activity following rapid remodeler depletion, we investigate the interplay between these enzymes and their impact on transcription in budding yeast. We show that many promoters are acted upon by multiple CRs that operate either cooperatively or in opposition to position the key transcription start site-associated +1 nucleosome. Functional assays suggest that +1 nucleosome positioning often reflects a trade-off between maximizing RNA Polymerase II recruitment and minimizing transcription initiation at incorrect sites.Finally, we show that nucleosome movement following CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity +1 nucleosomes maintain their positions. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.

Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.