Sequence determinants for promoter strength in the leuV operon of Escherichia coli

Abstract

The promoter for the leuV tRNA operon of Escherichia coli has been studied. Derivatives of this promoter were examined in vivo, fused to the cat gene or to the lacZ gene. When compared to other promoters, the leuV promoter was found to be at least three times stronger than the tyrT promoter (for the tyrT tRNA operon), or the lac promoter (trp::lac promoter fusion) and as strong as the P1,P2 promoter of the rrnB operon (a ribosomal RNA operon). Deletion analysis revealed that, while removal of sequences downstream from +11 (relative to the transcription start point) did not affect activity, removal of sequences upstream from -39 resulted in a ten-fold reduction in expression. Unlike rRNA operons which also display upstream activation, sequences responsible for this effect in the leuV promoter are separated into two regions, one between -76 and -47, and the other between -45 and -39. DNA fragments carrying the leuV promoter migrate aberrantly on polyacrylamide gels, a phenomenon usually associated with DNA bending. One sequence thought to be involved in bending is a TTTTT run centered around -71. Point mutations engineered at this T5 region resulted in a loss of activation but had no apparent effect on migration rate. Transcription efficiency of promoter derivatives was examined in vitro using supercoiled, relaxed, or linearized plasmids as templates. Upstream activation was observed only when using relaxed templates, although maximum activity was obtained using supercoiled forms. Insertion of the very efficient 16S transcription terminator between the leuV promoter and the cat gene resulted in barely detectable activities, indicating that no antitermination mechanism was present.