Lac Repressor

Lac repressor properties - The lac repressor was isolated in 1966 by Walter Gilbert and Benno Müller-Hill. The purified lac repressor is a tetramer, formed from four identical subunits, each with 360 amino acids (M r = 38,350). The protein binds isopropylthiogalactoside (IPTG, a synthetic inducer) with a Ka of about 106 M-1, and it binds nonspecifically to duplex DNA with a Ka of about 3 x 106 M-1. However, its specific binding at the lac operator is much tighter, with a Ka of 1013 M-1. Like RNA polymerase, lac repressor seeks its operator site by first binding to DNA at any site and then moving in one dimension along it. It moves either by sliding or by transfer from one site to another, when the two sites are brought next to each other on adjacent loops of DNA.

Lac repressor binding of operator - Control by the lac repressor is exceedingly efficient, particularly in view of the minute amount of repressor present in an E. coli cell. The i gene is expressed at a very low rate, to give about 10 molecules of repressor tetramer per cell. Although this corresponds to a concentration of only about 10-8 M, this value is several orders of magnitude higher than the dissociation constant, meaning that in a noninduced cell the operator is bound by repressor more than 99.9% of the time--hence, the very low levels of lac operon proteins in uninduced cells (less than one molecule per cell). Binding of inducer by lac repressor decreases the affinity of the repressor-inducer complex for operator by many orders of magnitude. Under these conditions, nonspecific binding of the repressor-inducer complex at other DNA sites becomes significant, so that in induced cells the operator is bound by the lac repressor less than 5% of the time.

The Repressor Binding Site - The DNA site bound by lac repressor has been analyzed by footprinting and methylation protection experiments. The lac operon operator's DNA sequence comprises 35 base pairs, including 28 base pairs of symmetrical sequence; that is, the sequence is identical in both directions (shaded in the diagram). Thus, the operator is an imperfect palindrome--imperfect because there are 7 base pairs that do not show this symmetry. The transcriptional start point is included within the repressor-binding sequence, as shown in the diagram. Figure 26.19 shows how the operator and promoter overlap, as determined by the regions of DNA protected by binding either repressor or RNA polymerase, respectively.

Looped structure - After the lac operon had been sequenced, two additional lac repressor- binding sites were located nearby, one centered at position -82, and one within the lacZ gene itself, at position +432. Both sites participate in lac operon regulation. Evidence indicates that a looped DNA structure is essential for complete repression, with the repressor contacting both the -82 and +11 sites. The tetrameric protein consists of two dimeric units, joined by a hinge region. Each dimer binds DNA separately, suggesting that the tetrameric protein binds to both the +11 and -82 sites, creating a DNA loop of 93 base pairs between them.

See also: Lactose Operon Regulation, cAMP Receptor Protein (CRP), RNA Polymerases

INTERNET LINKS:

> 1. The Lac Operon

> 2. Induction of the Lac Operon