Localization of the sequence-determined DNA bending center upstream of the streptokinase gene skc
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© Springer-Verlag 1996
O R I G I N A L PA P E R
Steffen Groß · Klaus Gase · Horst Malke
Localization of the sequence-determined DNA bending center upstream of the streptokinase gene skc
Received: 13 March 1996 / Accepted: 31 May 1996
Abstract DNA sequences upstream of the core promoter region of the streptokinase gene (skc) from Streptococcus equisimilis H46A increase skc transcription more than tenfold in vivo. This promoter upstream region contains a segment of intrinsically bent DNA, the precise location of which was determined experimentally by circular permutation analysis and theoretically by computer prediction. Electrophoretic analysis of circularly permuted upstream DNA fragments placed the bend center approximately at position –100 with respect to the major transcription initiation site of skc. This position was in excellent agreement with the center of maximum curvature predicted theoretically. Knowledge of the precise location of the bend center will be useful for future studies of the possible effect of DNA bending on skc transcription. Key words Streptococcus equisimilis · skc gene · DNA bending · Circular permutation analysis · Computer modeling Abbreviations BEND DNA Specific 500-bp DNA segment exhibiting intrinsic curvature · USR Promoter upstream region
Introduction Streptococcus equisimilis H64A, a human serogroup C strain, is a potent producer of the plasminogen activator streptokinase (Christensen 1945). This protein is exploited clinically for fibrinolytic therapy (GUSTO Angiographic Investigators 1993) and regarded as a virulence factor of the pathogenic streptococci that increases their invasiveness (Lottenberg et al. 1992; Malke et al. 1994;
S. Groß · K. Gase · H. Malke (Y) Institute for Molecular Biology, Jena University, Winzerlaer Strasse 10, D-07745 Jena, Germany Tel. +49-3641-657530; Fax +49-3641-657520 e-mail [email protected]
Gase et al. 1996). Streptokinase combines stoichiometrically with plasminogen to form a streptokinase-zymogen complex (Rodriguez et al. 1995) that then acquires an active site capable of converting other plasminogen molecules to the serine protease plasmin (Young et al. 1995). The streptokinase gene from H46A, skc (Malke et al. 1985), is abundantly transcribed as monocistronic mRNA (Mechold et al. 1993) from a complex promoter structure that we have examined recently by S1 nuclease mapping, reporter gene fusion, and directed mutagenesis (Gase et al. 1995; Gräfe et al. 1996). The functionally important sequences in the skc promoter region revealed by these studies include two overlapping core promoters (P1 and P2) arranged in tandem on opposite faces of the DNA helix, and a promoter upstream region (USR) necessary for full skc expression. In the homologous environment, P1 and P2 alone are extremely weak, and in the USR-less arrangement, only the combined core promoters have substantial activity. The USR stimulates only P1 (about 30fold) and the combination of P1 and P2 (about 15-fold). In contrast, in Escherichia coli virtually the entire promoter activity
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