Structure and Function in Escherichia coli of Plasmids Containing Pyrimidine/Purine-Biased Stretch Originated from the 5'-Flanking Region of the Basidiomycete ras Gene

Abstract

The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained. To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT [ras] and pBR-invCT [ras] were introduced into E. coli strains DM800 (ΔtopA gyrB225) and JM109 (topA⁺ gyrA96). In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)-coding strand and in pBR-invCT [ras] the complementary purine-rich sequence is on this strand. DNAs of pBR-CT [ras] and pBR-invCT [ras] isolated from DM800 were frequently cleaved with single-strand-specific Sl nuclease within the CT/AG sequence, showing the formation of extended open structure. Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT [ras] showed much higher levels of tetracycline resistance (Tc^r), while both strains carrying pBR-invCT [ras] showed clearly lower levels of Tc^r. pBR-CT [ras] and pBR-invCT [ras], however, conferred reduced activity of β-lactamase on DM800 and JM109. pBR-CT [ras] derivatives lacking the counterpart of the mirror repeat did not form the Sl-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tc^r and β-lactamase activity. The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.