Isolation and characterization of Zymomonas mobilis DNA fragments showing promoter activity in Escherichia coli = Escherichia coli에서 promoter 활성을 보이는 Zymomonas mobilis DNA 조각의 분리와 분석

Abstract

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 4-8 bps 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 (Delta G degrees) of -12.4 kcal/mol.