A whole-genome approach to identifying protein binding sites: promoters in Methanocaldococcus (Methanococcus) jannaschii.
We have adapted an electrophoretic mobility shift assay (EMSA) to isolate genomic DNA fragments that bind the archaeal transcription initiation factors TATA-binding protein (TBP) and transcription factor B (TFB) to perform a genome-wide search for promoters. Mobility-shifted fragments were cloned, tested for their ability to compete with known promoter-containing fragments for a limited concentration of transcription factors, and sequenced. We applied the method to search for promoters in the genome of Methanocaldococcus jannaschii. Selection was most efficient for promoters of tRNA genes and genes for several presumed small non-coding RNAs (ncRNA). Protein-coding gene promoters were dramatically underrepresented relative to their frequency in the genome. The repeated isolation of these genomic regions was partially rectified by including a hybridization-based screening. Sequence alignment of the affinity-selected promoters revealed previously identified TATA box, BRE, and the putative initiator element. In addition, the conserved bases immediately upstream and downstream of the BRE and TATA box suggest that the composition and structure of archaeal natural promoters are more complicated.
Li
Enhu
E
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Reich
Claudia I
CI
Olsen
Gary J
GJ
D019843:Archaeal Proteins / Q000378:metabolism; D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D020745:Genome, Archaeal; D017017:Methanococcus / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D035181:TATA-Box Binding Protein / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D019843:Archaeal Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors