1994 Volume 40 Issue 6 Pages 541-549
Promoter libraries of Zymomonas mobilis were generated in Escherichia coli using the CAT promoterless vector, pKK232-8. Among the 15 clones retained for further study, quantitative analysis of promoter strength revealed a 140-fold difference between the weakest and strongest promoter fragment. Nucleotide sequence determination and primer extension analysis were used to locate possible promoter-like sequences. Inspection of the DNA sequence surrounding the mRNA start-site, revealed regions similar to the E. coli sigma 70 consensus sequence (TTGACA-17bps- TATAAT), approximately 4-8bps upstream. Analogous to E. coli upstream activators, regions high in AT content (70-80%) and rich in static DNA bands, were found preceding the -35 hexamer. Only one promoter fragment clone (Z1) was found to carry an open reading frame preceded by a sequence resembling a ribosome-binding site with a free energy of SD-anti-SD binding (ΔG°) of -12.4kcal/mol.