Mutational Analysis of an Archaebacterial Promoter: Essential Role of a TATA box for Transcription Efficiency and Start-Site Selection in vitro

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 24; pp. 9509 - 9513
• Main Authors: Reiter, Wolf-Dieter, Hudepohl, Uwe, Zillig, Wolfram
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01-12-1990; National Acad Sciences
• Subjects: Archaea - genetics; Base Sequence; Biological and medical sciences; Chromosome Deletion; DNA; DNA probes; DNA, Ribosomal - genetics; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Nucleic acids; Nucleotides; Plasmids; Promoter regions; Promoter Regions, Genetic; Ribosomal DNA; RNA; RNA, Ribosomal, 16S - genetics; RNA, Ribosomal, 23S - genetics; Sulfolobus; TATA Box; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Molecular hybridization; Nucleotide sequence; Transcription; Archaeobacteria; Transcription promoter; Point mutation; Deletion; Bacteria; Molecular probe; Sulfolobus; Transcription initiation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.24.9509

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 defined 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). All 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 finding suggests a common evolutionary origin of these promoters consistent with the known similarities between archaebacterial and eukaryotic RNA polymerases.