BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 28. THE REGULATION OF GENE EXPRESSION IN THE PHENOTYPE

28.2. Discovery of the Regulatory Gene

The key to investigating The Mechanism of β-galactosidase induction was the discovery that, in the presence of all tested Inducers, the amounts of permease and transacetylase increased in direct proportion to The amount of β-galactosidase. Further progress was achieved through The Study of mutants. This demonstrated that β-galactosidase, permease, and transacetylase are encoded by three genes—designated $z$, $y$, and $a$, respectively—which are located consecutively. Mutants that lost The ability to synthesize one of these Proteins were successfully isolated. For example, the genotype $z^-y^+a^+$ indicates that a given mutant lacks β-galactosidase but possesses normal quantities of permease and transacetylase. Of greatest interest is a Class of mutants in which all three proteins are affected by the mutation. Such constitutive mutants synthesize large quantities of β-galactosidase, permease, and transacetylase in the complete absence of an inducer. François Jacob and Jacques Monod concluded that The rate of synthesis of these three proteins depends on a common element distinct from the genes encoding their sequences. The Gene for this common regulatory element was designated $i$. Inducible wild-type Bacteria have the $i^+z^+y^+a^+$ genotype, whereas lactose gene constitutive mutants have the $i^-z^+y^+a^+$ genotype.

How does the $i$ gene exert its effect on the rate of Synthesis of the proteins encoded by the $z$, $y$, and $a$ genes? The simplest hypothesis was that the $i$ gene encodes a cytoplasmic component termed a repressor, which is either entirely absent in $i^-$ Cells or inactive within them. This hypothesis was tested through a series of elegant genetic experiments using partially diploid bacteria carrying two sets of genes for the lactose region. One set was contained within the bacterial chromosome, while the second was introduced into The Cell via an F' sex factor during conjugation. For example, an $i^+z^-/Fi^-z^+$ diploid was constructed. In this diploid, the $i^+z^-$ genes reside on the chromosome, whereas the $i^-z^+$ genes reside on the episome. Is this diploid inducible or constitutive with respect to β-galactosidase? In other words, can the $i^+$ gene of the bacterial chromosome suppress the expression of the $z^+$ gene located on the episome? The experiment yielded a definitive result: the diploid is inducible rather than constitutive. An analogous result was obtained for the $i^-z^+/Fi^+z^-$ diploid. Consequently, the $i$ gene encodes a diffusible repressor.

28.3. The Operon: A Unit of Coordinated Genetic Expression

Building upon the experiments just described, Jacob and Monod postulated the Operon model to explain the Regulation of Protein Synthesis. The genetic elements of this model comprise a regulatory gene, an operator gene, and a set of structural genes (Fig. 28.3). The regulatory gene produces a repressor capable of interacting with the operator gene. It was subsequently established that the repressor is a protein. The operator gene is situated immediately adjacent to the structural genes it controls. The binding of the repressor to the operator gene prevents the METABOLISM/31.html">Transcription of the structural genes. The operator gene, together with the structural genes adjacent to it, is designated as an operon. In the case of the lactose operon, the $i$ gene serves as the regulatory gene, the $o$ gene as the operator gene, and the $z$, $y$, and $a$ genes as the structural genes. Furthermore, a promoter region (denoted by the symbol $p$) exists for the binding of RNA polymerase. This Transcription initiation site is located upstream of the operator gene. An inducer, such as isopropyl β-D-1-thiogalactopyranoside (IPTG), binds to the repressor, thereby disrupting its interaction with the operator gene. The $z$, $y$, and $a$ genes are subsequently enabled to undergo transcription. This process yields a single long RNA molecule encoding all three proteins (Fig. 28.4). An mRNA molecule that encodes more than one protein is referred to as a polycistronic (or polygenic) transcript.

Fig. 28.3. Map of the lactose operon and its regulatory gene. The map is not drawn to scale: the $p$ and $o$ regions are actually much smaller than the protein-coding Regions of the genes

Fig. 28.4. Schematic diagram of the lactose operon in the repressed (A) and induced (B) states

28.4. The lac Repressor is a Tetrameric Protein

The Isolation of the lactose operon repressor (the lac repressor) exploited its capacity to bind IPTG. Walter Gilbert and Benno Müller-Hill demonstrated that the lac repressor is a protein that binds to DNA containing the lac operon while failing to bind to any other DNA. As predicted, IPTG inhibits the binding of the lac repressor to the lac operator DNA. Wild-type E. coli cells contain only about ten molecules of the lac repressor. Difficulties arose during the purification of the repressor because it accounts for merely 0.001% of the total cellular protein. However, $i^{ ext{q}}$ mutants exist that apparently possess a more efficient promoter for the $i$ gene. These mutants synthesize vastly greater amounts of the lac repressor. The yield of the lac repressor is enhanced still further through The Use of transducing phages carrying the lac region. E. coli cells infected with such a phage contain approximately 20,000 repressor molecules (roughly 2% of the total protein), providing an excellent Starting Material for the isolation of the lac repressor.

Fig. 28.5. Electron micrograph of the lac repressor bound to DNA containing the lac operator

The repressor is a tetramer composed of identical subunits with a mass of 37 kDa, each possessing a single inducer-binding site. The dissociation constant for IPTG is approximately 10^-6 M. The repressor binds to the operator with extraordinary strength and rapidity. The dissociation constant for the repressor-operator complex is approximately 10^-13 M. Such exceptionally high affinity is necessitated by the fact that a wild-type E. coli cell harbors only a handful of repressor molecules. The association rate constant is strikingly high at 7 • 10⁹ M^-1 • s^-1. This indicates that the repressor locates the operator site by diffusing along the DNA molecule (one-dimensional search) rather than searching for it within the aqueous medium (three-dimensional search)¹.

¹ Recent studies have demonstrated that the situation is considerably more complex: the repressor's search for the operator encompasses both mechanisms and is profoundly dependent on the ionic COMPOSITION OF THE medium. — Transl. note.