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The Lac Repressor Protein

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The Lac Repressor Protein Proc. Nat. Acad. Sci. USA Vol. 71, No. 3, pp. 593-597, March 1974 The lac Repressor Protein: Molecular Shape, Subunit Structure, and Proposed Model for Operator Interaction Based on Structural Studies of Microcrystals (electron microscopy/x-ray diffraction/protein-DNA interaction) THOMAS A. STEITZ, TIMOTHY J. RICHMOND, DAVID WISE, AND DONALD ENGELMAN Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 Communicated by Frederic M. Richards, October 31, 1973 ABSTRACT Electron microscopic and powder x-ray We would like to suggest another model for repressor- diffraction studies of small crystals of the lac repressor pro- tein provide evidence on its molecular shape and subunit operator interaction that is consistent with the asymmetric structure which in turn suggests a possible mode of repres- molecular shape of the repressor derived from x-ray and elec- sor-operator interaction. The crystals are probably ortho- tron microscopic studies reported here. This model allows all rhombic space group P2221 with unit cell dimensions of four subunits of the repressor to interact with the operator. a = 140, b = 91, c = 117 A. This tetrameric protein appears It proposes 222 symmetry for the repressor, as has been found rather asymmetric, having approximate molecular dimen- for all sions of 140 A by 60 A by 45 A. The dumbbell shape of the other tetrameric proteins of known structure (16). projected molecular outline observed in the electron Finally, it is consistent with the partial 2-fold axis found re- micrographs can be explained by assuming that the sub- lating the nucleotide sequence of the operator by Gilbert and units are related by 222 symmetry and are placed at the coworkers (13). corners of a plane rectangle. We propose a model for re- pressor- operator interaction in which the DNA binds to Crystallization the repressor with its long axis aligned with that of the repressor and with its 2-fold axis coincident with a twofold The lac repressor protein used in these experiments was iso- axis of the repressor. lated at Harvard in Prof. K. Weber's laboratory by Dr. T. Platt and TAS (14). Crystals were obtained by dialyzing The control of DNA transcription into messenger RNA is best repressor protein against 40 mMNl phosphate, pH 7.0, and understood in the case of the lactose operon of Escherichia coli 10 1iNI isopropyl-f-D-thiogalactoside. The crystals are thin (1-7). Transcription of the lactose operon by RNA polymerase needles up to 0.2-mm long. Although the crystals obtained is prevented by the specific binding of the lac repressor protein thus far are too small for single crystal x-ray diffraction (150,000 molecular weight) to the operator locus. Induction analysis, we have obtained electron micrographs of negatively of 0-galactosidase by inducers, such as isopropyl-f-D-thio- stained crystals (Figs. 1 and 2) and powder x-ray diffraction galactoside, has been explained by the binding of the inducer photographs (Fig. 4). to the repressor which causes the repressor to dissociate from the DNA allowing transcription to proceed (1, 3, 6). We have Electron microscopic studies initiated structural studies on the lac repressor protein to From negatively stained electron micrographs of these re- elucidate the nature of the ol)erator-repressor interaction pressor crystals (Figs. 1 and 2) we have determined two unit- as well as the allosteric transition produced by inducer cell dimensions, the lattice symmetry of one projection and binding. the approximate size of two dimensions of the tetramer. In The interaction of the tetrameric lac repressor protein with the most ordered part of the electron micrograph, as judged DNA poses some interesting structural problems. Does the by the resolution of the optical diffraction pattern, two unit- repressor recognize double-stranded DNA (8, 9)? Does it cell dimensions were measured as 95 A and 115 A. Possible locally denature the operator and bind to single-stranded DNA errors in magnification and, thus, in cell dimensions are esti- (10)? Or does it bind to a region of the DNA that assumes a mated to be + 10%. The crystallographic symmetry elements cloverleaf structure (11, 12)? At least two models of repressor- observed are a glide line parallel to the needle axis, 2-fold operator interaction have been proposed to explain how the axes perpendicular to the micrograph, and mirror lines per- four identical subunits of the repressor, which has four binding pendicular to the long axis of the crystal. Hence, the symmetry sites for inducer (3), can bind to one operator and make use of this projection is probably pgm, which suggests that the of all four subunits. Muiller-Hill and colleagues (9) suggest minimum crystal point group symmetry is D2. The simplest that the four subunits are arranged in a superhelix so that interpretation of the micrograph is that each tetrameric each subunit can bind to a DNA sequence that is repeated molecule appears in this projection as a dumbbell or waisted four times in the operator. Sobell (12), expanding on a model ellipsoid (Fig. 1 right; Fig. 2). Although these dumbbell shapes proposed by Gierer (11), proposes that the operator folds intq might result from a more complex superposition of molecules, a cloverleaf structure that possesses approximate 4-fold sym- we have not found another interpretation consistent with all metry relating its arms, and that the repressor likewise has the data. a 4-fold axis relating its four subunits, each of which contains Optical diffraction patterns (Fig. 3) of electron micrographs an identical DNA binding site. of these crystals show reflections out to 15 A along the crystal 593 Downloaded by guest on September 28, 2021 594 Biophysics: Steitz et al. Proc. Nat. Acad. Sci. USA 71 (1974) FIG. 1. Electron micrographs of lac repressor crystals. (left) An electron micrograph of a negatively stained lac repressor crystal taken using an Hitachi HU-11E at about 51,000 times magnification at 100 kV. The crystal was crosslinked with 0.1% glutaraldehyde, placed on a carbon-coated Formvar film grid, and stained with 0.2% uranyl acetate. Square encloses one unit cell containing four molecules as shown in the right of Fig. 5. (right) A partial real-space photographic average of the micrograph shown at the left. This real- space averaging of the projected contents of several unit cells was accomplished by very precise translation of the print by exact multiples of the unit-cell vector parallel to the long crystal axis and taking a multiple exposure photograph through a photographic enlarger, a method used by Labaw and Davies (20). Four to six translations produced an image that could not be improved by additional averaging. The most interesting part of the micrograph is to the left, where the unit-cell boundaries are clearest, suggesting we are viewing down the long a axis of the crystal. In this part of the photograph one can see white dumbbell- or waisted ellipsoid-shaped objects each of which corresponds to the projected density of one tetrameric lac repressor molecule and has dimensions of about 45 i by 60 A. The b axis is horizontal and the c axis is vertical in these micrographs. Note that the pgm symmetry relating the dumbbell-shaped molecules is clearly visible, suggesting that the structural detail was not introduced by the translational averaging. The part of the crystal to the right which shows less detail is thought to be twisted, giving a view oblique to the a axis. axis and 20 A0 across the crystal. The glide line results in in part on the two-cell dimensions obtained from the electron extinction of reflections 1 = 2n + 1. While the diffraction micrograph, the weak 91-A powder reflection is indexed as patterns of different areas of the micrograph vary in resolu- the (010) reflection and the strong 59-A spacing is indexed as tion and intensity distribution, those patterns extending to the (002) reflection; the (002) is also a strong reflection in the the highest resolution have quite similar intensity distribu- optical diffraction pattern (Fig. 3). tions. Unit cell dimensions, crystal packing, X-ray powder diffraction and molecular shape The most important result from the powder diffraction pattern Combining these data from electron micrographs and x-ray is that the largest spacing in these crystals is 140 A (Fig. 4). diffraction photographs, we conclude that the unit cell dimen- This reflection at 140 A spacing is best indexed as the (100) sions are 140 A by 91 A by 117 A with a = = y = 90°. reflection; attempts to index this reflection as the (200), These cell parameters yield a unit-cell volume of 1.5 X 106 A3. (110), (101), etc. predict reflections in the 140- to 90-A spacing If the unit cell contains four tetrameric molecules as the micro- regions which are not in fact observed. Thus, the third unit- graphs suggest, then the cell would also contain about 50% cell dimension, which is not observed in the electron micro- solvent or have a Vm of 2.5 A3 per dalton, the average value graph, is 140 A. Other peaks are observed in the powder pattern observed for protein crystals (15). at 90.8 A, 69.5 A, 58.8 A, 48.6 A, 39.2 A, and at higher angles. Since all three cell dimensions are different and since the Indexing of these peaks is not completely unambiguous be- micrograph shows a minimum symmetry of three perpendicu- cause of overlap of expected reflections. However, relying lar 2-fold (or 2-fold screw) axes, an orthorhombic crystal class Downloaded by guest on September 28, 2021 Proc. Nat. -Acad. Sci. USA 71 (1974) Structural Studies of lac Repressor Crystals 595 140 A r- c6 69.5 A- f"qI\ 9-4 4 58.8 A 1.2 A 21 0 "0.005 0.01 0.015 0.02 0.025 0.03 1/d (k') FIG.
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