Enhancer-dependent transcription involving the promoter specificity factor ⁵⁴ is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, ⁵⁴-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of ⁵⁴ promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by ⁵⁴ were evident, indicating loss of rpoN (encoding ⁵⁴) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual ⁵⁴-dependence in vivo not readily correlated with conventional ⁵⁴ binding-sites.