Amy Gimlen

The Lactose Operon, introduced by Jacob and Monod, is the classical model for gene regulation. It is the model for how structural genes are transcribed or repressed depending on the conditions within the cell. When lactose is not present in the cell the repressor is bound tightly to the operator region of the DNA and the lac Z gene (which codes for B-galactosidase), lacY (which codes for lactose permease), and lac A (which codes for transacetylase) are not transcribed. When lactose is present the repressor disassociates from the operator and the genes are transcribed. Control of the lac operon does not only depend on the association or disassociation of the repressor, cyclic AMP also plays a regulatory role. At high levels of cyclic AMP the CAP protein (cyclic AMP-dependent catabolite gene activator protein) raises the level of transcription, by raising the affinity of RNA polymerase for the promoter.

This exhibit will show three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer IPTG, and the lac repressor complexed with a 21-bp symmetric operator DNA. These structures show the conformation of the lac operon in both the induced and repressed states, will give information about the lac repressor, and how the repressor interacts with DNA and functions together with CAP to regulate the lac operon.

The lac repressor is a protein of 360 amino acids that forms a homotetramer. It has five distinct fragments: four NH2-terminal fragments and a COOH-terminal tetrameric core. The NH2-terminal fragments, each 60 residues, bind in a specific manner to the operator. The COOH-terminal tetrameric core binds the inducer.

The lac repressor monomer has four functional units: the NH2-terminal headpiece, the hinge region, a sugar binding domain and a COOH-terminal helix. The headpiece which binds to the DNA, contains a helix-turn-helix motif (HTH), this creates a small, compact globular domain that is hydrophobically rich due to two alpha helices joined by a turn to the third helix. The hinge connects the DNA binding domain to the core of the repressor. This segment in the absence of DNA allows the headpiece to move independently of the core of the repressor. When DNA is bound the hinge becomes ordered in an alpha helix, which causes specific interactions with the operator on the DNA and it orients the headpieces. The core, inducer binding domain, is composed of two subdomains, six-stranded parallel beta sheets sandwiched between four alpha helices. Three linkers link the subdomains. The COOH-terminus contains a short segment of 11 residues followed by a COOH-terminal alpha helix that contains two leucine heptad repeats. When four of theses COOH-terminal helices associate the tetramer oligomerization domain is formed.

The lac repressor tetramer should rather be considered as a dimer of dimers. Its structure is essentially a pair of tethered dimers. It creates a repressor that is roughly V-shaped that is not fixed precisely.

The inducer molecule, IPTG, binds to the repressor where the NH2-terminal and the COOH-terminal subdomains interface. The binding of the inducer to the repressor reduces the affinity of the lac repressor for the DNA. The IPTG molecule is pseudo symmetric, this fact is what allows the molecule to interact with the repressor molecules in two different ways. The two ways vary due to only in the contact with the sugar. The repressor can either form three or four hydrogen bonds to the IPTG hydroxyl groups. The orientation with four hydrogen bonds involves the side chain of Asn 246 with O2 of the galactoside, Arg 197 with O3 and O4, and Asp 149 with the O6 position. This sugar binding pocket has a hydrophobic surface formed by Leu 73, Ala 75, Pro 76, Ile 79, Trp 220, and Phe 293, and an isopropyl group of the IPTG within van der Waals contact of Trp 220. The three hydrogen bond orientation is seen in the lac repressor core fragment.

The lac operon has three lac repressor recognition sites in a stretch of 500-bp. They are at the positions of three operator sites, O1, O2, and O3. O3 lies with in the lac I gene, which is 93 bp upstream of O1, which lies within the promoter. O2 lies 401 bp downstream of O1, within the lac Z gene. The operators have a nearly dyad symmetry. O1 is more symmetric than O2 and O3, therefore it binds the tightest. The lac repressor binds ten times greater to the palindrome of the left half of the operator. In the repressor-DNA complex, each repressor tetramer is bound to two independent, symmetric DNA operators. Therefore each dimer binds to one operator site. The primary site of interaction is the HTH motif, which fits snuggly into the major groove of the DNA. Due to very limited resolution crystal data the exact interaction cannot be determined, but the residues Leu 6, Tyr 17, Gln 18, Ser 21, Arg 22, and His 29 are close to the DNA, and their side chains most likely form the base pair interactions with the operator in the major groove of the DNA. There is a secondary interaction with the bases in the minor groove of the DNA, the pair of leucine residues in the hinge helices make direct contact. The pair of leucine residues acts to pry open the minor groove of the DNA.

The binding of the repressor to the 21-bp symmetric operator distorts the conformation of the DNA so that it bends away from the repressor with an approximately 60 angstrom radius of curvature. The distortions in the DNA are localized to the center of the operator where there is a kink of about 45 degrees that is seen in the minor groove. The DNA was seen to unwind locally about 50 degrees. These distortions are what allow the hinge helices to interact with the minor groove.

There are two distinct structural rearrangements of the monomer and the dimer, corresponding to the induced and repressed states. The conformation of the repressor monomer, uncomplexed, is extremely similar to the repressor in the presence of IPTG. In contrast the difference is about five times greater from the uncomplexed repressor to the operator complexed repressor. The repressor adopts a conformation in the presence of the operator that differs from both the unliganded and the inducer bound forms. The NH2- terminal and the COOH-terminal subdomains in the induced form and the repressed form only differ by a small hinge motion. This hinge motion alters the positions of the NH2-terminal subdomains within the dimer. In the induced state of the repressor the hinge helices interactions are interrupted, it frees up the DNA binding HTH domains, and reduces the affinity of the repressor for the operator.

The tetramer is essentially tethered dimers that move together and apart. A single lac repressor tetramer can bind two independent pieces of operator DNA, which is either 93 or 401 bp apart. The conformation that the continuous DNA must take is what is called the repression loop. There are two hypotheses that are possible for creating the repression loop. Two subunits of the tetramer can bind to the primary operator and the other dimer can associate to the ancillary operator, this is specific for the O1 and O3 repression loop. Or, two free repressor dimers bind to separate operators and a loop occurs when the dimers associate into the tetramer. Either way the repressor acts as a clamp bringing two operators that are separated in a linear sequence closer together in space. CAP also plays a role in the repression loop, which seems paradoxical, but it induces a 90 degree bend over about 30bp between the two operators. The regression loop model also positioned the RNA polymerase binding site on the inside of the loop, which prevents the polymerase from accessing the promoter. There is also a O1 to O2 repression loop.

M. Lewis, G. Chang, N. Horton, M, Kercher, H. Pace, M. Schumacher, R. Brennan, P. Lu, " Crystal Structure of the Lactose Operon Repressor and Its Complexes with DNA and Inducer," in Science 271,1247 (1996)