Lac repressor
Encyclopedia
The lac repressor is a DNA-binding protein which inhibits the expression
of gene
s coding for proteins involved in the metabolism
of lactose
in bacteria. These genes are repressed when lactose
is not available to the cell, ensuring that the bacterium only invests energy in the production of machinery necessary for uptake and utilization of lactose when lactose is present. When lactose becomes available, it is converted into allolactose
, which inhibits the lac repressor's DNA binding ability. Loss of DNA binding by the lac repressor is required for transcriptional activation of the operon.
. This blocks RNA polymerase from binding, and so prevents transcription
of the mRNA coding for the Lac proteins. When lactose is present, allolactose binds to the lac repressor, causing an allosteric change in its shape. In its changed state, the lac repressor is unable to bind tightly to its cognate operator. This effect is referred to as induction, because it induces, rather than represses, expression of the metabolic genes. In vitro, Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a commonly-used allolactose mimic which can be used to induce transcription of genes being regulated by lac repressor.
DNA binding occurs via an N-terminal helix-turn-helix
structural motif
and is targeted to one of several operator DNA sequences (known as O1, O2 and O3). The O1 operator sequence slightly overlaps with the promoter, which is thought to therefore prohibit binding by RNA polymerase
thereby inhibiting expression of the operon. Additionally, because each tetramer contains two DNA-binding subunits, binding of multiple operator sequences by a single tetramer induces DNA looping.
by Walter Gilbert
and Benno Müller-Hill in 1966. They were able to show, in vitro
, that the protein
bound to DNA containing the lac operon, and released the DNA when IPTG
was added. (IPTG is an allolactose analog
.) They were also able to isolate the portion of DNA bound by the protein by using the enzyme
deoxyribonuclease
, which breaks down DNA. After treatment of the repressor-DNA complex, some DNA remained, suggesting that it had been masked by the repressor. This was later confirmed.
These experiments confirmed the mechanism of the lac operon, earlier proposed by Jacques Monod
and Francois Jacob
.
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
of gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s coding for proteins involved in the metabolism
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
of lactose
Lactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...
in bacteria. These genes are repressed when lactose
Lactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...
is not available to the cell, ensuring that the bacterium only invests energy in the production of machinery necessary for uptake and utilization of lactose when lactose is present. When lactose becomes available, it is converted into allolactose
Allolactose
Allolactose is a disaccharide similar to lactose. It consists of the monosaccharides D-galactose and D-glucose linked through a β1-6 glycosidic linkage instead of the β1-4 linkage of lactose. It may arise from by the occasional transglycosylation of lactose by β-galactosidase.It is an inducer of...
, which inhibits the lac repressor's DNA binding ability. Loss of DNA binding by the lac repressor is required for transcriptional activation of the operon.
Function
The lac repressor (LacI) operates by binding to the major groove of the operator region of the lac operonLac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
. This blocks RNA polymerase from binding, and so prevents transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of the mRNA coding for the Lac proteins. When lactose is present, allolactose binds to the lac repressor, causing an allosteric change in its shape. In its changed state, the lac repressor is unable to bind tightly to its cognate operator. This effect is referred to as induction, because it induces, rather than represses, expression of the metabolic genes. In vitro, Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a commonly-used allolactose mimic which can be used to induce transcription of genes being regulated by lac repressor.
Structure
Structurally, the lac repressor protein is a homo-tetramer. The tetramer contains two DNA binding subunits composed of two monomers each (sometimes called "dimeric lac repressor"). These subunits dimerize to form a tetramer capable of binding two operator sequences. Each monomer consists of four distinct regions:- an N-terminal DNA-binding domain (in which two LacI proteins bind a single operator site)
- a regulatory domain (sometimes called the core domain, which binds allolactose, an allosteric effector molecule)
- a linker that connects the DNA-binding domain with the core domain (sometimes called the hinge helix, which is important for allosteric communication)
- a C-terminal tetramerization region (which joins four monomers in an alpha-helix bundle)
DNA binding occurs via an N-terminal helix-turn-helix
Helix-turn-helix
In proteins, the helix-turn-helix is a major structural motif capable of binding DNA. It is composed of two α helices joined by a short strand of amino acids and is found in many proteins that regulate gene expression...
structural motif
Structural motif
In a chain-like biological molecule, such as a protein or nucleic acid, a structural motif is a supersecondary structure, which appears also in a variety of other molecules...
and is targeted to one of several operator DNA sequences (known as O1, O2 and O3). The O1 operator sequence slightly overlaps with the promoter, which is thought to therefore prohibit binding by RNA polymerase
RNA polymerase
RNA polymerase is an enzyme that produces RNA. In cells, RNAP is needed for constructing RNA chains from DNA genes as templates, a process called transcription. RNA polymerase enzymes are essential to life and are found in all organisms and many viruses...
thereby inhibiting expression of the operon. Additionally, because each tetramer contains two DNA-binding subunits, binding of multiple operator sequences by a single tetramer induces DNA looping.
Discovery
The lac repressor was first isolatedProtein purification
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or...
by Walter Gilbert
Walter Gilbert
Walter Gilbert is an American physicist, biochemist, molecular biology pioneer, and Nobel laureate.-Biography:Gilbert was born in Boston, Massachusetts, on March 21, 1932...
and Benno Müller-Hill in 1966. They were able to show, in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
, that the protein
bound to DNA containing the lac operon, and released the DNA when IPTG
IPTG
Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent.This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon...
was added. (IPTG is an allolactose analog
Analog (chemistry)
In chemistry, a structural analog , also known as chemical analog or simply analog, is a compound having a structure similar to that of another one, but differing from it in respect of a certain component. It can differ in one or more atoms, functional groups, or substructures, which are replaced...
.) They were also able to isolate the portion of DNA bound by the protein by using the enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
deoxyribonuclease
Deoxyribonuclease
A deoxyribonuclease is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Thus, deoxyribonucleases are one type of nuclease...
, which breaks down DNA. After treatment of the repressor-DNA complex, some DNA remained, suggesting that it had been masked by the repressor. This was later confirmed.
These experiments confirmed the mechanism of the lac operon, earlier proposed by Jacques Monod
Jacques Monod
Jacques Lucien Monod was a French biologist who was awarded a Nobel Prize in Physiology or Medicine in 1965, sharing it with François Jacob and Andre Lwoff "for their discoveries concerning genetic control of enzyme and virus synthesis"...
and Francois Jacob
François Jacob
François Jacob is a French biologist who, together with Jacques Monod, originated the idea that control of enzyme levels in all cells occurs through feedback on transcription. He shared the 1965 Nobel Prize in Medicine with Jacques Monod and André Lwoff.-Childhood and education:François Jacob is...
.
External links
- More information on the lac repressor molecule on protein database
- Lac Repressor in Proteopedia.