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Nucleic Acids Research, 1989, Vol. 17, No. 13 5017-5028
© 1989

ENZYMOLOGY

Micrococcal nuclease as a probe for bound and distorted DNA in lac transcription and repression complexes

Li Zhang and Jay D. Gralla

Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90024, USA

To whom correspondanace should be addressed

Received April 17, 1989. Revised June 30, 1989. Accepted June 30, 1989.

Micrococcal nuclease (MNase) is used to probe the structure of transcription and repression complexes at the lac regulatory region in vitro. Both the lac operator, 01, and the pseudo-operator, 03, are found to be protected from MNase digestion by the lac repressor on supercoiled DNA, and hypersensitive sites appear on both strands around nucleotide (nt) —26 between 01 and O3. This hyperreactive site is coincident with the site of the DNA kink shown previously to form within a loop caused by simultaneous repressor binding to 0, and 03. MNase hypersites are also observed both upstream from cAMP receptor protein (CRP) and downstream from bound RNA polymerase in open promoter complexes. In both open and closed complexes the binding of polymerase partially protects the backbone from MNase attack. Catabolite activator protein is shown to be required for both closed and open complex formation. Taken together with previous footprinting data, the results suggest that lac transcription complexes involve DNA bent towards a protein core consisting of RNA polymerase and catabolite activator protein.


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