IN-VIVO DEFINITION OF AN ARCHAEAL PROMOTER

We have used a plasmid-based transcriptional reporter system to examine the transcriptional effects of 33 single point mutations in the box A region (TATA-like sequence) of the Haloferax volcanii tRNA(Lys) promoter. The most pronounced effects on transcriptional efficiency were found when the nucleotides corresponding to the TATA-like region were altered. Promoters with wild-type or higher levels of transcriptional activity conformed to the general archaeal box A consensus, 5'-T/CTTAT/AA-3'. The preference for a pyrimidine residue in the 5' position of this region and the exclusion of guanine and cytosine in the next four positions in the 3' direction are defining characteristics shared by all efficient archaeal promoters. We have also observed that replacement of a 10-nucleotide purine-rich sequence, located 5' of the H. volcanii tRNA(Lys) box A element, completely abolished transcription from this promoter. These data show that the H. volcanii tRNA(Lys) promoter is dependent on tno separate, and essential, sequence elements. The possible functions of these sequences, in view of the recent descriptions of eucaryal-like transcription factors for Archaea, are discussed.