| A transcriptional promoter under the positive control of both araC protein and cAMP receptor protein was cloned from E. coli K12 chromosomal DNA using a promoter-cloning plasmid, pKB260. The pKB260 tetracycline genes can only be expressed by a foreign promoter inserted at the unique plasmid Eco R1 site. Thus the search for promoter sequences under control of araC was reduced to a search for recombinant plasmids expressing arabinose inducible tetracycline resistance.;The cloned ara promoter was shown to be under araC control both in vitro and in vivo, and under cAMP receptor protein control in vitro. The promoter was found to be located at one end of a 330 base pair fragment of DNA by restriction enzyme mapping and in vitro transcription. The DNA sequence of this fragment was determined. While a computer assisted search of this sequence revealed several Pribnow box sequences, no other striking homologies to the published DNA sequence of the araCBAD control region were found. From the length of the ara transcript and from the orientation of transcription, one of these Pribnow sequences was identified as being immediately adjacent to the probable origin of transcription.;Portions of the first 500 base pairs of the putative araFG message sequence were compared to the published amino acid sequence of arabinose binding protein, whose gene, araF, is the first known gene in the araFG operon. No similarities were found between the arabinose binding protein amino acid sequence and translations in all three reading frames of portions of the putative message sequence, indicating that there may be an additional gene preceding araF in this operon.;The cloned DNA fragment carring the ara promoter was found to hybridize to a plasmid, from the Clarke-Carbon E. coli plasmid bank, which carries the araFG operon. Thus it is reasonably certain that the cloned ara promoter is the araFG promoter. |
