Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1 , is highly induced under oxygen-limiting conditions. A DNA gyrase inhibito r, novobiocin, was previously shown to block bop gene induction and suggest ed that DNA supercoiling mediates transcriptional induction. A region of no n-B structure was found 3' to the TATA box within an 11 bp alternating puri ne-pyrimidine sequence (RY box), which correlated to both increased DNA sup ercoiling and transcriptional induction. Here, saturation mutagenesis of th e RY box region has been used to show that single-base substitutions of A(r )G either 23 or 19 bp 5' to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mut agenesis of the region 5' to the TATA box showed its involvement in DNA sup ercoiling modulation of transcription, defined the 3' end of the upstream a ctivator sequence (UAS) regulatory element, and ruled out the requirement f or a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited trans cription indicating that the UAS element does not function as a transcripti onal enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regu latory and transcription factors in bop promoter activity.