Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter

Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minim al bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS), The mutagenized bop promoter and th e wild-type structural gene and transcriptional terminator were inserted in to a shuttle plasmid capable of replication in the halophilic archaeon Halo bacterium sp, strain S9, Active promoters were isolated by screening transf ormants of an orange (Pum(-) bop) Halobacterium mutant for purple (Pum(+) b op(+)) colonies on agar plates and analyzed for bop mRNA and/or bacteriorho dopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a )Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of th e transcription start site, A putative UAS, 5'-ACCcnactagTTnG-3', located 5 2 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement o f the TATA box and UAS for bop promoter activity.