Elisabeth Bertrand-Burggraf

Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number

Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced. These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+). Similar induction was observed with the product of another plasmid gene, beta-lactamase. The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.

Kinetic studies of the modulation of ada promoter activity by upstream elements.

We have determined the kinetics of initiation of transcription of the wild‐type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M‐1 and 1.7 x 10(‐2) s‐1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild‐type sequence with an up‐mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (‐0.05), leading to a 12‐fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)

STRUCTURE OF UVRABC EXCINUCLEASE UV-DAMAGED DNA COMPLEXES STUDIED BY FLOW LINEAR DICHROISM - DNA CURVED BY UVRB AND UVRC

The interaction between UvrABC excinuclease from Escherichia coli and ultraviolet light‐(UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence off effector ATP. The change was specific for UV‐damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV‐damaged DNA. The structural modification of DNA, promoted by the (UvrA2‐UvrB) complex probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.

Fast abortive initiation of uvrA promoter in a supercoiled plasmid studied by stopped-flow techniques

In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped‐flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 μl), and the short dead time (∼0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3‐fold. Nevertheless, it does not change significantly the product K B k 2, which is indicative of promoter strength and shows that uvrA is an ‘association‐limited’ promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.