Mutational analysis of an archaebacterial promoter: essential role of a TATA box for transcription efficiency and start-site selection in vitro.

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Abstract
By using a recently developed in vitro transcription assay, the 16S/235 rRNA-encoding DNA promoter from the archaebacterium Sulfolobus, sp. B12 was dissected by deletion and linker substitution mutagenesis. The analysis of 5'' and 3'' deletion mutants defined a core promoter region between positions -38 and -2 containing all information for efficient and specific transcription. Further characterization this region by linker substitution mutagenesis indicated two sequence elements important for promoter function-one located between positions -38 and -28 (distal promoter element) and the other one located between positions -11 and -2 (proximal promoter element). The distal promoter element encompassed the TATA-like "box A" element located approximately 26 nucleotides upstream of the majority of transcription start sites in archabacteria (Archaeobacteria). All mutations within this box A motif virtually abolished promoter function. Complete inactivation of the proximal promoter element was dependent on extensive mutagensis; this element is not conserved between arachaebacterial promoters except for a high A+T content in stable RNA gene promoters from Sulfolobus. 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 selection were dependent on a conserved TATA-like sequence centered approximately 26 nucleotides upstream of the initiation site, a situation unlike that of eubacterial promoters but resembling the core structure of most eukaryotic RNA polymerase II (and some RNA polymerase III) promoters. This finding suggests a common evolutionary origin of these promoters consistent with the known similarities between arachaebacterial and eukaryotic RNA polymerases.