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Contacts Between Escherichia Coli RNA Polymerase and Thymines In Proc. NatI. Acad. Sci. USA Vol. 76, No. 7, pp. 3233-3237, July 1979 Biochemistry Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter (Escherichia coli genetic control/RNA polymerase-promoter interaction/bromouracil substitution/photochemical probe/ RNA nucleotidyltransferase) ROBERT B. SIMPSON* Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Communicated by Walter Gilbert, April 23, 1979 ABSTRACT I have identified those 5 positions of thymines enine on double-stranded DNA, other probes with different in the lac UV5 promoter that lie close to bound Escherichia coli specificities are needed to get a complete picture of the crucial RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl- promoter elements. transferase, EC 2.7.7.6). Although ultraviolet irradiation of DNA interaction between RNA and with 5-bromouracil substituted in place of thymine normally I have probed the polymerase cleaves the DNA at the bromouracils, a protein bound to the a strong promoter by using a photochemical method developed DNA can perturb these cleavages at those locations at which by Ogata and Gilbert (22) that identifies 5 positions of thymine the protein lies close to the bromine. In the lac promoter most that are close to a bound protein. Ultraviolet irradiation of the of these contacts lie in three regions. Four contacts lie in the DNA, with 5-bromouracil substituted in place of thymine, region where transcription initiates; four lie in the "Pribnow displaces the bromine and leaves an activated uracilyl radical box," which is located about 10 base pairs upstream from the in the major groove of the DNA helix. Normally, this free initiation site; and three more lie in the "-35 region," located about 35 base pairs upstream from the initiation site. The radical seizes a hydrogen atom from the sugar on the 5' side, "Pribnow box" and the "-35 region" are regions whose se- destroying the ribose ring and breaking the DNA at that point quences are partially conserved between promoters and in (23). However, the presence of a bound protein can alter the which most promoter mutations are located; thus, contacts in cleavage at locations where the protein is close enough to the these two regions probably represent sites of sequence-specific free radical to react with it. The experiment, then, is to monitor recognition by RNA polymerase. this cleavage in the presence and absence of bound RNA Which features of a promoter direct RNA polymerase polymerase at each bromouracil in the promoter. (nucleosidetriphosphate:RNA nucleotidyltransferase, EC The lac UV5 promoter mutant is a strong promoter charac- 2.7.7.6) to initiate RNA synthesis? DNA sequence determina- terized by a rapid association with RNA polymerase resulting tion of promoters and promoter mutants provides a partial in a stable complex (8, 24, 25). Unlike the wild-type promoter, answer to this question. When the sequences of promoters are it is not catabolite repressed: RNA polymerase action does not compared, two regions of prominent homology have been ob- require activated cyclic AMP receptor protein (26). Thus this served: the "Pribnow box" and the "-35 region" located about promoter is a simple object for studies in vitro of a chromosomal 10 and 35 base pairs, respectively, upstream from the start site promoter. of transcription (1-4). Furthermore, almost all promoter My results identify points of contact between RNA poly- mutations are located within these regions (5-12). merase and the lac UV5 promoter. The locations within the Another approach examines different portions of the pro- promoter sequence of the contact points are nonrandom. Their moter for their accessibility to various probes in the presence dispositions stress the importance of particular regions within or absence of RNA polymerase. For example, RNA polymerase the promoter. One region covers the site where transcription bound to any of several promoters protects about 40 base pairs begins. The other two regions correspond well to the location of DNA from endonuclease attack (1, 2, 13, 14). Although of promoter mutations, the contacts inferred from the dimethyl polymerase in the resulting complex can initiate correctly and sulfate probe of this promoter by Johnsrud (21), and the posi- synthesize a transcript about 20 bases long, the 40 base pair tions of homologies revealed when available promoter se- fragment does not rebind the polymerase, showing that this quences are compared. fragment lacks some information required for promoter rec- MATERIALS AND METHODS ognition. And, in fact, enzymatic digestion with exonuclease (15) or restriction endonucleases (5, 9, 16-19) suggests that RNA Polymerase. RNA polymerase was purified from roughly 65 base pairs of DNA are required and that the ap- Escherichia coli K cells (Grain Processing Co., Muscatine, IA) proximate boundaries of a promoter are located about 45 base by the method of Burgess and Jendrisak (27). Then chroma- pairs upstream and 20 base pairs downstream from the site of tography on single-stranded DNA-agarose as suggested by initiation. A finer probe is the chemical attack on DNA by di- Lowe et al. (28) yielded RNA polymerase with a complete methyl sulfate (20), which gives a more detailed picture of the complement of the a subunit. Polyacrylamide gel electropho- interaction because the chemical probe is much smaller than resis in sodium dodecyl sulfate showed that the protein was the enzymes that degrade DNA. This probe shows that contacts more than 95% pure. in one promoter extend from the point of initiation to at least Bromouracil-Substituted DNA. Promoter DNA was isolated 38 base pairs upstream (21). Because the dimethyl sulfate at- from the tetracycline-resistant plasmid pLJ3 constructed by tacks only the N-7 group of guanine and the N-3 group of ad- Johnsrud (21). All manipulations of recombinant DNA were done at the P1 level of containment. To increase the incorpo- The publication costs of this article were defrayed in part by page ration of exogenous bromouracil, the plasmid was carried in a charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate *Present address: Department of Microbiology, University of Wash- this fact. ington, Seattle, WA 98195. 3233 Downloaded by guest on September 24, 2021 3234 Biochemistry: Simpson Proc. Nati. Acad. Sci. USA 76 (1979) mutant requiring thymine (FMA 10 from F. Ausubel; W3102, the DNA, denaturing the products, and separating the resulting lacking restriction enzyme). The cells were grown in M9 me- fragments by size with gel electrophoresis. The distance be- dium plus casamino acids (29) supplemented with thymine at tween the label and the break identifies the location of the 2 Aig/ml and tetracycline at 10 ,g/ml. At an ODsso of 1.2, the break. Thus, the autoradiograph of the gel reveals a series of plasmid was amplified by the addition of chloramphenicol to bands corresponding to breaks at successive bromouracils in the 170 ,gg/ml. At the same time bromouracil was added to a con- labeled strand of the fragment. If RNA polymerase is bound centration of 50 ,g/ml. After incubation overnight at 370C, to the fragment during the irradiation, the intensity of specific the plasmid DNA was isolated as described by Tanaka and bands changes. My interpretation is that these bands correspond Weisblum (29) with one modification. Because DNA in which to bromouracils in the immediate vicinity of the bound pro- bromouracil has substituted for thymine has a density greater tein. than that of unsubstituted DNA, the final density of the ethi- Fig. 1 illustrates the results of such an experiment. Labeled dium bromide/CsCl gradient was increased to 1.65 g/ml. From restriction fragments were irradiated in the absence or presence this gradient, I isolated a broad band that represented heavily of increasing amounts of RNA polymerase. The fragments were substituted supercoiled plasmid DNA. labeled at the 5' terminus of the "top strand" (as portrayed in Johnsrud (21) described the isolation from this plasmid of Fig. 4). The figure shows the autoradiograph of such a gel. In restriction fragments containing the promoter 32P-labeled at lane c, each band represents ultraviolet-induced cleavage at a the 5' end of one or the other strand. The "top" and "bottom" bromouracil in the labeled strand. The pattern of the same DNA strands (defined in Fig. 4) are labeled on fragments of 117 and cleaved at the bromouracils by chemical means (lane b) iden- 168 base pairs, respectively. Restriction enzymes Hae III and tifies the irradiation products. The patterns are displaced by EcoRI were generous gifts from A. Jeffrey and L. Johnsrud, one base with respect to each other because chemical attack respectively. D'Andrea and Haseltine (30) have published the decomposes the sugar attached to the bromouracil (unpublished deoxynucleotide sequence of these fragments. data), whereas irradiation destroys the sugar that is the 5' Ultraviolet Irradiation. The ultraviolet light source was a neighblr of the bromodeoxyuridine (23). The bases are num- 500-W medium-pressure mercury lamp (Hanovia), jacketed bered with respect to the startpoint of transcription (see Fig. with a chilled circulating solution of 1 M NiSO4 and 0.2 M 4). As Ogata and Gilbert (22) observed, the ultraviolet-induced CuSO4. The NiSO4 absorbs most of the infrared radiation while cleavage is not uniform throughout the DNA fragment but the CuSO4 absorbs most ultraviolet light with wavelengths less appears to be sequence specific. As a result, some bands are too than 300 nm. This spectrum of radiation is preferentially ab- sorbed by the bromouracil, because between 300 and 315 nm the absorption coefficient of bromodeoxyuridine is 10 to 100 a d e times greater than that of thymidine (31). The number of breaks in the DNA is proportional to the time of irradiation.
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