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

Abstract

By using a recently developed in vitro transcription assay, the 16S/23S 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 defmed a core promoter region between positions -38 and -2 containing all information for efficient and specific transcription. Further characterization of this region by linker substitution mutagenesis indicated two sequence elements important for promoter function-one located 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 encompassed the TATA-like "box A" element located approximately 26 nucleotides upstream of the majority of transcription start sites in archaebacteria (Archaeobacteria). Al mutations within this box A motif virtually abolished promoter function. Complete inactivation of the proximal promoter element was dependent on extensive mutagenesis; this element is not conserved between archaebacterial 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 rmding suggests a common evolutionary origin of these promoters consistent with the known similarities between archaebacterial and eukaryotic RNA polymerases.Based on molecular data, archaebacteria (Archaeobacteria) comprise a group of prokaryotic microorganisms phylogenetically distinct from eubacteria and eukaryotes (1-3). Many archaebacteria are characterized by extreme habitats believed to resemble the environmental conditions during the early evolution of life, leading to the suggestion that they represent an ancient group of organisms (1, 2).Transcription in archaebacteria has primarily been investigated on the level of DNA-dependent RNA polymerases. These studies indicate a single transcribing enzyme displaying a subunit complexity similar to the three eukaryotic nuclear enzymes (4). The characterization of archaebacterial transcription signals has been confined to nucleotide sequence comparisons, revealing two conserved sequence elements, a "box A" motif [consensus TTTA(A or T)A] centered approximately 26 nucleotides upstream of the transcription start site and a "box B" motif [consensus (A or T)TG(A or C)] containing the transcription start site on the central guanosine residue or on another pu...

Role of MerH in mercury resistance in the archaeon Sulfolobus solfataricus

“…Thus, an alternative strategy was required and TATA box mutagenesis was used to identify the main mer operon promoter. S. solfataricus promoters studied in vitro are typically T/A-rich octameric sequences while weaker noncanonical promoters are G/Crich (Bell et al, 1999;Reeve, 2003;Reiter et al, 1990). In the data presented here, in vivo replacement of both merRp-TATA and merHp-TATA with G/C-rich octameric sequences resulted in reduced promoter activity and noticeable physiological effects while similar manipulations had no effect on a putative merAp.…”

Regulation of expression of the arabinose and glucose transporter genes in the thermophilic archaeon Sulfolobus solfataricus

“…5). It was mapped to a G that was six nucleotides upstream of the translational start codon (ATG) of the araS gene and 29 nucleotides downstream of a canonical TATA-box (Reiter et al 1990). Attempts to detect a transcription start side for the araT gene failed with this method.…”

A Second Terminal Oxidase in Sulfolobus acidocaldarius

“…possible promoter sequence (TTTAAA; Reiter et al, 1990) is present between positions -25 and -30 from the 5' end of the mRNA (nucleotide 135 in Fig. 4).…”