MUTATIONAL ANALYSIS OF AN ARCHAEBACTERIAL PROMOTER - ESSENTIAL ROLE OF A TATA BOX FOR TRANSCRIPTION EFFICIENCY AND START-SITE SELECTION INVITRO

By using a recently developed in vitro tran tion assay, the 16S/23S rRNA-encoding DNA promoter the archaebacterium Sulfolobus sp. B12 was dissected by and linker substitution mutagenesis. The analysis of 5′ 3′ deletion mutants defined a core promoter region be positions -38 and -2 containing all information for efficient and specific transcription. Further characterization of region by linker substitution mutagenesis indicated two sequence elements important for promoter function - one loaded between positions -38 and -25 (distal promoter element) and the other one located between positions -11 and -2 proximal promoter element). The distal promoter element uncompassed the TATA-like "box A" element located approximately 26 nucleotides upstream of the majority of transcription start sites in archaebacteria (Archaeobacteria). All mutations within this box A motif virtually abolished promoter tion. Complete inactivation of the proximal promoter ment was dependent on extensive mutagenesis; this element conserved between archaebacterial promoters except for a high A+T content in stable RNA gene promoters from folobus. Mutants containing insertions or deletions between the distal and proximal promoter elements were only slightly affected in their transcription efficiency but displayed a shift in their major initiation site, retaining an essentially fixed distance between the distal promoter element and the transcription start site. Thus, efficient transcription and start-site tion were dependent on a conserved TATA-like sequence red approximately 26 nucleotides upstream of the initiation site, a situation unlike that of eubacterial promoters but bling the core structure of most eukaryotic RNA poly ase II (and some RNA polymerase III) promoters. This ng suggests a common evolutionary origin of these promoters consistent with the known similarities between archaebacterial and eukaryotic RNA polymerases.