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Specific protection of nucleotides

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Gene, 18 (1982) 247-255 Elsevier Biomedical Press 241 Spe cific protection of nucleotides in the lac operator from DMS methylation and DNase I nicking by crude ba cte rial cell extracts (Escherichia coli; DNA footprinting; regulatory proteins) Gary B. Ruvkun, Venkatesan Sundaresau and Frederick M. Ausubel Cellular and Developmental Group, Department of Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138 (U.S.A.) (Received January 26th, 1982) (Accepted March 22nd, 1982) SUMMARY Crude bacterial cell extracts prepared from an Escherichia coli fucIq strain were shown to protect specific nucleotides in the fuc operator from methylation by dimethyl sulfate (DMS) or digestion by DNase I, whereas no protection was observed using extracts prepared from a nearly isogenic lucZ_ strain. These experiments show that it is not necessary to use purified regulatory proteins in experiments designed to localize sequences on DNA which interact with proteins. Therefore, crude cell extracts should be useful in DNA “footprinting” experiments to define regions of DNA which bind to unknown regulatory proteins. INTRODUCTION Transcriptional control in prokaryotes is gener- ally mediated by regulatory DNA binding proteins which recognize specific nucleotide sequences near genes. In “classic” studies of bacterial genes and operons such as luc, c1, and cro (Watson, 1977), detailed genetic analyses provided models of gene regulation which were subsequently verified and refined by in vitro biochemical analyses using purified regulatory proteins and specific DNA templates. Regulatory regions on DNA templates Abbreviations: bp, base pairs; CAP, catabolic activation pro- tein; dH,O, distilled water; DMS, dimethyl sulfate; IPTG, isopropyl-B-D-thiogalactoside; PME, /3-mercaptoethanol; UREB, universal restriction enzyme buffer. were delimited by sequencing an RNA copy of the DNA fragment protected from DNase I digestion by a purified regulatory protein (Gilbert and Maxam, 1973) and, more recently, by identifying specific nucleotides protected from methylation by DMS (Gilbert et al., 1976) or digestion by DNase I (Galas and Schmitz, 1978; Johnson et al., 1979) upon binding to purified regulatory proteins (“footprinting”). The synergism between genetic and biochemical analysis is exemplified in studies of the fuc operon. The lac promoter/operator region was initially defined, localized, and a model for its role in regulation derived from genetic mapping studies and from complementation analysis of luc muta- tions (Beckwith, 1978). Subsequent experiments used purified lac repressor, E. coli RNA poly- merase, and purified DNA fragments containing 0378-l 119/82/0000-0000/$02.75 0 1982 Elsevier Biomedical Press248 the /UC promoter and operator to confirm that /UC repressor and RNA polymerase bind to adjacent, overlapping DNA sequences, and that each inter- acts with a specific set of nucleotides (Reznikoff and Abelson, 1978). An important feature of these latter experiments was the mapping of single base-change regulatory mutations to the protected regions which verified the involvement of these sequences in regulation. In contrast to “classical” biochemical-genetic studies in which extensive genetic analyses precede in vitro biochemical analyses, the most efficient use of recombinant DNA techniques involves the adoption of the reverse experimental strategy. In both prokaryotic and eukaryotic species, many genes can now be readily cloned; however, very few, if any, regulatory mutations are available and, in most cases, there are no simple genetic selec- tions to obtain such mutants. In the absence of regulatory mutations, the mechanism by which these genes are regulated must initially be de- termined via biochemical analysis. To circumvent the problem that regulatory proteins have not been identified and purified for most cloned genes, we sought to determine whether crude cell extracts could be used in conjunction with chemical and enzymatic DNA protection techniques (Gilbert et al., 1976; Galas and Schmitz, 1978; Johnson, 1979) to detect changes in the binding to DNA tem- plates of regulatory proteins in crude extracts pre- pared from cells expressing the cloned genes at different levels. This use of crude cell extracts would be useful because it directly identifies DNA sequences involved in binding regulatory proteins, highlights specific nucleotides for site-directed mutagenesis, and can indicate whether the control is positive or negative. Crude cell extracts have been used with purified DNA templates to map regions of the DNA which are transcribed (Travers et al., 1970) and trans- lated (DeVries and Zubay, 1967), and to study transcriptional control by comparing transcripts synthesized by extracts prepared from cells in dif- ferent states or to which various purified regula- tory proteins have been added. This use of cell extracts utilizes transcription as an assay for regu- latory protein binding and does not directly look at the DNA-protein interactions. As a model system, we have performed chemi- cal (Gilbert et al.. 1976) and enzymatic (Galas and Schmitz. 1978) protection analysis of the E. co/i /UC, operator using cell extracts prepared from /ucZq and fuel bacterial strains, rather than purified luc repressor protein. We report here that sonicated cell extracts prepared from lucIq cells protect the same nucleotides in the fuc operator as protected by purified luc repressor (Gilbert and Muller-Hill. 1967) whereas extracts prepared from /UC/ cells do not protect any nucleotides on the luc operator. MATERIALS AND METHODS (a) Escherichia co/j strains JMlOl = A(luc pro) BI supE[F’truD36 proAB /ucIq ZAM15] was the IucZq strain used tb make the cell extracts. The strain was


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