An operon is a collection of linked genes under common, coordinate control. The enzyme beta-galactosidase hydrolyzes the disaccharide lactose to the monosaccharides glucose and galactose. The concentration of beta-galactosidase is very low in cells normally (basal level); but when lactose is the sole carbon source, the concentration of the enzyme is elevated markedly (a process called induction). After all the lactose is metabolized, beta-galactosidase eventually returns to a very low level in the cell. The study of the induction of beta-galactosidase has been made easier by the use of a non-metabolized inducer isopropyl beta-thiogalactoside, also called IPTG (page 998), and a non-inducing substrate, called Xgal, which turns BLUE when hydrolyzed by beta-galactosidase. The lactose operon is the most intensely studied genetic regulatory system and is extraordinarily useful in genetic engineering.
The lactose operon
of E. coli consists of three structural
genes under coordinate
control (Figure
26.17). Transcription
commences at a promoter
(lacP) to the left of lacZ and transcribes a 5,200
nucleotide messenger RNA molecule (mRNA), ending at a terminator
beyond lacA. The three genes on the polygenic (polycistronic)
messenger are separately translated and the products are beta-galactosidase
(which cleaves lactose into galactose and glucose), permease (required
for transporting lactose across the cell membrane), and transacetylase
(which has a known, but not essential, enzymatic activity).
The operon is under the control of the adjacent lacI gene,
encoding the lactose repressor.
The repressor is a regulatory
gene. In the absence of allolactose, the inducer
of the lac operon, the repressor tetramer binds to the
lac operator
(lacO) and prevents RNA polymerase from transcribing the
operon. However, when allolactose is present it binds to the repressor
this prevents repressor from binding to lacO and permits
RNA polymerase to bind to lacP and to initiate transcription.
The lacI gene is transcribed constitutively
from its own promoter.
Regulatory mutants affecting the control of the lactose operon could be identified. There were two classes of constitutive mutants. One class are operator constitutive (oc) mutants: repressor cannot bind to the mutant operator site. oc mutants are cis-dominant; that is, they control only their adjacent operon, not an operon on another DNA molecule (such as a plasmid). The other class are i- mutants: repressor monomers cannot associate to form tetramers and thus cannot bind to the normal operator site. Normal repressors can form tetramers and can thus bind to the normal operator site. This means that normal repressor is dominant in trans to the mutant repressor (or, vice-versa, the mutant is trans-recessive). The table shows the genetic behavior of these mutants (the normal gene for beta-galactosidase is z+ and the mutant gene unable to make beta-galactosidase is z-).
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| Type of Control |
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| Negative (lac repressor) |
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| Positive (CRP protein) |
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The lac operon is actually MORE COMPLICATED than shown in the textbook. There are really three repressor binding sites: O1, O2, and O3 (Figure AF). All three binding sites are required for maximal repression (1,000-fold range of lac mRNA).
Regulation of the lac operon by repressor is called negative control (Figure P1; see also an older view of repression in Figure 26.21 on page 1002). The lac operon is also under positive control by CRP (or cAMP Receptor Protein; also known as CAP or catabolite activator protein). CRP or CAP is now thought to be bound to its lac binding site at all times (even during repression).
During induction, the inducer (either the natural inducer, allolactose, or the synthetic inducer, IPTG, binds to the lac repressor. Inducer-bound repressor does not bind to operator sites. This allows RNA polymerase to bind to the promoter and start transcribing the lac operon.
CRP (cAMP Receptor Protein; also known as CAP) is a dimer (Figure 26.29) and has a DNA-binding structural element called helix-turn-helix (HTH); only when complexed with at least one cAMP will CRP bind to site in the lac promoter (in fact, when two cAMP bind to dimeric CRP, the binding of CRP to target DNA is reduced 10-fold); CRP binding bends DNA more than 90° (Figure 26.22); CRP binding increases the formation of the closed complex of RNA polymerase (increases RNA polymerase binding to the promoter) by more than 20-fold (the lac promoter has a "poor" -35 box and a "poor" -10 region; some promoter up-mutants (mutant promoters that "work" better) that make the -35 box and/or the -10 region more like the consensus become independent of CRP activation). The spacing between the CRP site and the RNA polymerase site is important: insertion of 5 bp eliminates positive control by CRP, but insertion of 10 bp restores positive control to about 20% (thus, the orientation between CRP and RNA polymerase is thought to be important; that is, they must bind to the same "side" of the DNA molecule). CRP "touches" the alpha subunit of RNA polymerase (Figure X). Some rpoD (sigma factor) mutants also overcome the "need" for CRP positive control.
Figure AE shows footprinting of CRP and the lac repressor. When CRP binds to its site, repressor binding at O3 is shifted upsteam by 6 bp. This puts CRP and repressor on opposite sides of the DNA. Figure 26.19 shows the sequences that bind CRP and repressor.
Figure AG shows that tetrameric repressor binds to O1 and O3 to form a DNA loop (with the aid of CRP which helps to bend the DNA).
Several important points:
E. coli grown on glucose do not metabolize lactose (as well as several other sugars) until all of the glucose in the medium is exhausted. This is called catabolite repression. It once was thought that catabolite repression was mediated by the levels of cAMP in the cell which, in turn, affected the binding of CRP to its binding site in the lac operon. However, cAMP levels are the same in cells grown on glucose alone or on lactose alone. Glucose represses the lac operon of E. coli by prohibiting the entry of lactose though an occasionally present lac permease molecule. In the presence of glucose there is never enough lactose inside the uninduced cells to be transformed by the very few molecules of beta-galactosidase into the true inducer allolactose.