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Regulation of Bacterial Gene Activity Help (page 2)

By — McGraw-Hill Professional
Updated on Aug 23, 2011

Negative, Inducible Control

The classic example of negative control by way of an inducible operon is the lactose operon of E. coli. β-galactosidase is an enzyme with dual functions. Its primary function is to catabolize lactose to glucose and galactose. Its secondary function is to convert the 1-4 linkage of glucose and galactose (in lactose) to a 1-5 linkage in allolactose. This enzyme is not normally present in high concentrations when lactose is absent from the cell's environment. Shortly after adding lactose to a medium in which glucose is absent, the enzyme begins to be produced. A transport protein called galactoside permease is required for the efficient transport of lactose across the cell membrane. This protein also appears in high concentration after lactose becomes available in the medium. The wild-type lactose operon (Fig. 10-11) consists of a regulatory gene (lacI) and an operon containing a promoter sequence (lacP), an operator locus (lacO), and three structural genes for β-galactosidase (lacZ), permease (lacY), and transacetylase (lacA; an enzyme whose function in lactose metabolism remains unresolved).

Regulation of Bacterial Gene Activity

Mutations at each of these loci have been found allowing for detailed understanding of the functions of each of these genes.

Promoter Alleles

p + = wild-type promoter; normal affinity for RNA polymerase

p = mutant promoter cannot bind RNA polymerase; none of the structural genes in the lactose operon are transcribed

p s = increased affinity for recognition by RNA polymerase; elevates the transcriptional level of the operon; s = "super promoter".

p i cr = affects the CRP-cAMP binding site to reduce the level of expression of lactose operon genes below 10% of wild type; i cr = insensitive to catabolite repression

Operator Alleles

O + = in the absence of repressor, this operator "turns on" the structural genes in its own operon; i.e., the lacZ+ and lacY+ alleles in the same segment of DNA (cis position) can produce proteins; this operator is sensitive to the repressor; i.e., repressor will "turn off" the synthetic activity of the structural genes in the lactose operon

O c = a constitutive operator that is insensitive to repressor and permanently "turns on" the structural genes in the lactose operon

Galactosidase Alleles

Z + = makes β-galactosidase if its operon is "turned on" or "open"

Z = a missense mutation that makes a modified, enzymatically inactive product called lacCZ protein

Z ns = results in the destruction of the polycistronic message down stream from the mutation so that there is no expression of any of the downstream lactose operon genes (a polar mutation); ns = nonsense

Permease Alleles

Y + = makes β-galactoside permease if its operon is "turned on"

Y = no detectable permease is formed regardless of the state of the operator; probably a nonsense mutation

Regulator Alleles

I + = makes a diffusible repressor protein that inhibits synthetic activity in any o+ operon in the absence of lactose; in the presence of lactose, repressor is inactivated

I = a defective regulator that is unable to produce active repressor due to a nonsense or missense mutation

I s = makes a "super repressor" that is insensitive to lactose and inactivates any O+ operon

There is some overlap in the promoter and operator sites of the lac system; in some other operons the operator locus may be totally embedded in the promoter. The regulatory gene constitutively produces a repressor protein at low levels because it has an inefficient promoter. Its synthesis is unaffected by the level of lactose in the cell. The normal promoter of the lac operon, by contrast, binds RNA polymerase very efficiently. In the absence of lactose (noninduced conditions), an active repressor protein (produced by lacI) binds to the operator. RNA polymerase can neither bind to the promoter nor "read through" the operator sequence because repressor protein occupies that region. Hence, transcription of all three structural genes in the lac operon is prevented.

When lactose is present (induced conditions), it is transported inefficiently into the cell because only a few molecules of permease would normally be present. Inside the cell, some of the lactose would be converted to allolactose by β-galactosidase. Allolactose is the inducer of the lac operon. It binds to the repressor protein and causes a conformational change in the protein that alters the site by which it binds to the operator. This conformational change in a protein as a consequence of binding to another molecule is called an allosteric transformation. The allolactose-repressor complex can no longer bind to the operator, and it falls off the DNA. RNA polymerase can now read through the operator to transcribe the structural genes in the operon. The increased amount of permease now transports lactose across the membrane in large quantities, and the sugar is then digested by β-galactosidase. When lactose becomes depleted from the medium, newly synthesized repressor proteins will not be coupled with allolactose, so they can bind to the operator and shut off transcription of the structural genes in the operon. Furthermore, allolactose can reversibly bind to repressor protein, so that under low levels of lactose in the cell allolactose would tend to dissociate from repressor-allolactose complexes. Even when the lac operon is repressed, occasionally the repressor protein will diffuse from the operator momentarily. RNA polymerase may then be able to "sneak" past the open operator and synthesize a molecule of polycistronic mRNA, thus accounting for the very low levels of permease and β-galactosidase that are normally present in the cell. Bacterial mRNA molecules have a very short half-life (only a few minutes), so synthesis of proteins stops very soon after a cell is repressed. Proteins, on the other hand, are much more stable, but they would be diluted out with each subsequent cell division.

EXAMPLE 10.8 Bacteria of genotype I+ O+ Z+ Y+ grown on media devoid of lactose will produce neither galactosidase nor permease because I+ makes repressor substance that inactivates the O+ operator and "turns off " the synthetic activity of structural genes Y+ and Z+in its own operon.

EXAMPLE 10.9 Partial diploids can be produced in bacteria for this region of the chromosome. Cells of the genotype I O+ Z+ Y / I+ Oc ZY+ will produce the lacCZ protein constitutively (i.e., either with or without the presence of lactose inducer) because the allele Oc permanently "turns on" the genes in its operon (i.e., those in cis position with Oc). β-Galactosidase will be produced only inductively because in the presence of lactose (inducer), the diffusible repressor substance from lacI+ will be inactivated and allow the structural gene lacZ+ in cis position with the operator O+ to produce enzyme.

The wild-type operon of the regulatory gene (lacI) in the lactose system consists of just a promoter (pi) and the structural gene for the repressor protein (I+). Its wild-type promoter is very inefficient, and only a few molecules of lac-repressor protein exist in the cell. In the operons of most regulatory genes in other systems, however, an operator locus is adjacent to its promoter, and autoregulation is possible. The repressor proteins made by these operons bind to their own operators to terminate transcription when the concentrations of their respective repressor molecules are elevated.

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