Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.