Text Assignment: Chapter 8, Pages 216-229.
Major Concepts
Symbol substitution: Please not that "ß-" has been used throughout these notes to indicate "beta-" because of lack of the appropriate Greek letter beta in html. This characteris actually German, but if it does not come out looking vaguely like a beta, make sure that your browsers are set for the iso8859-1 character set (called Western [Latin-1] on some browsers).
Operons: An operon consists of a group of coordinately controlled genes that are all related to a particular cellular function. Operons occur primarily in prokaryotic organisms. Typically, the genes are physically adjacent to one another and all controlled from a single upstream promoter. A single polycistronic transcript is generated that contains coded information for translation of several proteins. An operator locus immediately adjacent to the promoter controls whether or not the operon is transcribed. Regulatory proteins bind to the operator to either repress or induce transcription. The activities of the regulatory proteins are altered by the presence or absence of various types of small molecules within cells. For example, in cells that lack glucose but have lactose, the genes controlled by the operator for the lac-operon are actively transcribed, which enables the cell to use lactose as an energy source. Expression of all of the genes in the operon is turned on or off simultaneously. Their coding sequences are all contained within a single polycistronic mRNA, whose transcription is under the control of the lac operator.
lac-operon: The lac operon consists of three structural (protein coding) genes whose products are involved in the utilization of lactose. ß-galactosidase (encoded by the lacZ gene) is responsible for the hydrolysis of lactose into galactose and glucose. ß-galactosidase also converts some lactose into allolactose, which plays a critical role in the induction of the lac operon. ß-galactoside permease (encoded by the lacY gene) is a protein that is required for transport of lactose into the cell. The third gene product, ß-galactoside acetyltransferase (encoded by the lacA gene), does not appear to be directly involved in lactose metabolism, but is thought to be involved in detoxification of related substances.
The lac operon is under dual control, repressed by the
presence of glucose and induced by the presence of lactose when
glucose is absent. A low level of expression is maintained constituitively,
providing a priming effect for induction. When induction is acheived,
there is a sharp increase in transcription of the polycistronic
message which codes for all three proteins.
Operator: The primary operator site (O1) is located right at the transcriptional start site (figure 8.3). A related site (O3) is located just upstream, and a third site (O2) is located downstream, embedded within the ß-galactosidase coding sequence. In the absence of protein interactions, the operator sites have no effect on transcription. However, a lac-repressor protein, coded by the nearby lacI locus, attaches firmly to the O1 site and somewhat less strongly to the O3 site. This causes the DNA to form a loop, which prevents the initiation of transcription (figure 8.5a).
Induction by allolactose: The lac-repressor protein, which is a homotetramer, has four binding sites for allolactose. When those sites become occupied, the protein undergoes a change in shape (allosteric modification), which causes it to dissociate from the operator site, allowing transcription to proceed (figure 8.5b). Thus allolactose induces the lac operon by a process of de-repression. In order for lactose to induce the operon, there must already be present a low level of permease to get the lactose into the cell and a low level of ß-galactosidase to convert the lactose to allolactose. Mutants that totally lack either the permease or ß-galactosidase cannot be induced by lactose.
Non-metabolized inducer: One of the problems encountered when using lactose or allolactose to induce the lac operon is that as soon as the enzymes of the operon are induced, they begin to destroy the inducer. One way around this problem is to use isopropyl-thio-ß-galactoside (IPTG), which is a potent inducer that is not metabolized. In addition, IPTG passes readily through cell membranes, eliminating the need for active permease in order to obtain induction.
Operator Mutation: The primary operator (O1)
is a specific palindromic DNA sequence that is recognized by
the lac-repressor protein. If this sequence changes
(mutates) to a sufficient degree, the repressor can no longer
bind to it. This type of operator mutation is denoted by Oc.
The "c" stands for constituitive, since this type
of operator mutation allows the operon to be consituitively transcribed
(always on). Operator mutations act in a cis-dominant mode,
such that they are not overcome by introducing a normal copy of
the gene in a merozygote.
Repressor mutations: Mutations in the LacI gene
that cause the repressor
protein to be unable to bind the operator (denoted I-)
will always be recessive when trans to a wild-type copy
of the gene, whose product will bind to both operators. Repressor
protein mutations that are unable to bind allolactose (denoted
IS) will remain bound to the operator locus, and will
be dominant in trans.
Structural gene mutations: A mutation that blocks the activity of ß-galactosidase will prevent the conversion of lactose to allolactose. Induction of the operon by lactose will be blocked, but externally-supplied allolactose will still serve as an inducer. The induced operon will not generate a functional ß-galactosidase, but the other two enzymes will be induced normally. A mutation that blocks the activity of the permease will block (or greatly impair) all induction unless artificial means are employed to get lactose into the cell.
Control of lac operon by glucose: There is an indirect negative control of the lac operon by glucose. The cell prefers to use glucose as its primary energy supply as long as it is available. As glucose levels fall, intracellular levels of cyclic adenosine monophosphate (cAMP) begin to rise. When this happens, an intracellular protein called Catabolite Activator Protein (CAP) binds to the cAMP and becomes a positive acting regulator of the lac operon, binding upstream from the -35 promoter sequence (see figures 8.13 and 8.7).
Dual control of lac operon: In order to achieve a high level of induction it is necessary 1) for the CAP site to be occupied by a CAP/cAMP complex, and 2) for the lac operator site to not be occupied by the repressor protein. The description of these controls can easily get confused. Glucose indirectly represses the lac operon by keeping cAMP levels low and blocking active induction by the CAP/cAMP complex. Allolactose induces the operon by binding to the repressor protein, causing it to release the operator, resulting in de-repression of the operon..
The trp-operon, which we will be examining in the next lecture,
is even more complex, so be sure to gain a full understanding of
the lac-operon as a starting point as quickly as possible.