Abstract: The predominant organizational theme by which the transcription machinery and chromatin regulators are positioned within promoter regions or throughout genes in a genome is largely unknown. We mapped the genomic location of diverse representative components of the gene regulatory machinery in Saccharomyces cerevisiae to an experimental resolution of <40 bp. Sequence-specific gene regulators, chromatin regulators, mediator, and RNA polymerase II were found primarily near the downstream border of the "-1" nucleosome, which abuts against the ~140 bp nucleosome-free promoter region (NFR). General transcription factors TFIIA, -B, -D, -E, -F, -H were located near the downstream edge of the NFR. The -1 nucleosome dissociated upon Pol II recruitment, but not upon recruitment of only TBP and TFIIB. The position of many of sequence-specific regulators in promoter regions correlated with the position of specific remodeling complexes, potentially reflecting functional interactions. Taken together the findings suggest that the combined action of activators and chromatin remodeling complexes remove the -1 nucleosome after the pre-initiation complex (PIC) has partially assembled, but before or concomitant with Pol II recruitment. We find PIC assembly, which includes Pol II recruitment, to be a significant rate-limiting step during transcription, but that additional gene-specific rate-limiting steps associated with Pol II occur after recruitment.