Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput datasets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif datasets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well-defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks. Display omitted •An underlying logic distinguishes NF-κB promoter binding from enhancer binding•NF-κB and IRF3 collaborate via diverse mechanisms to activate key inflammatory genes•Genes induced most rapidly and transiently by lipid A invariably support SRF binding Stringent analyses of nascent transcript RNA-seq, ChIP-seq, and transcription factor binding motif datasets associated with inflammatory gene induction uncover the extent to which unique mechanisms regulate individual genes with key biological functions and allow a mechanistic understanding of transcriptional control at a genome-wide level.