Transcriptional analysis of the Escherichia coli bio operon

Using primer extension, two in vivo transcription start points (tsp) were identified for rightward transcription of the Escherichia coli biotin operon, at nucleotides (nt) +20 and +29. The strongest leftward transcript begins at +9, with a tenfold less abundant transcript starting at +3. The activity of segments cloned into promoter probe vectors locates the major leftward promoter between nt +1 and +105, as expected for the +9 tsp. Although the activity of a chromosomal operator is reduced about 300-fold by point mutation in either arm of the palindrome extending from nt -20 to +20, either a half-operator segment or a full operator bearing the same point mutation in one arm is substantially repressed when cloned into pKB2000, as though cellular location strongly affects the operator's affinity for the repressor.