Major Concepts
Text Assignment: Chapter 18, pages 522-529.
Symbol substitution: Please not that "b-" has been used throughout these notes to indicate "beta-" because of lack of the appropriate Greek letter beta in html.
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 determines when the operon is actually actively 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 thelac-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 operator.
lac-operon: The lac operon consists of three genes whose products are involved in the utilization of lactose. b-galactosidase (encoded by the lacZ gene) is responsible for the hydrolysis of lactose into galactose and glucose. b-galactosidase also converts some lactose into allolactose, which plays a critical role in the induction of the lac operon. b-galactoside permease (encoded by the lacY gene) is a protein that helps to transport lactose into the cell. The third gene, b-galactoside acetyltransferase (encoded by the lacA gene) plays an indirect role which is not completely clear.
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 operator site is located right at the transcriptional
start site. In the absence of protein interactions, the operator
has no effect on transcription. However, a lac-repressor
protein, coded by the nearby lacI locus, attaches firmly
to the operator locus and prevents the initiation of transcription.
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. 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 b-galactosidase
to convert the lactose to allolactose. Mutants that totally lack
either the permease or b-galactosidase cannot be induced by lactose.
Operator Mutation: The operator is a specific 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 that result in the repressor
protein being unable to bind the operator locus (denoted I-)
will always be recessive when in 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 b-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 b-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 figure 18.6).
Dual control of lac operon: In order to achieve a high level of induction it is necessary that 1) the CAP site be occupied by a CAP/cAMP complex, and 2) the lac operator site is empty. 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 locus.
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.