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The ''lac'' repressor (LacI) is a DNA-binding protein that inhibits the expression of
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
s coding for
proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
involved in the
metabolism Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run ...
of
lactose Lactose is a disaccharide sugar synthesized by galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from ' (gen. '), the Latin word for milk, plus the suffix ' ...
in bacteria. These genes are repressed when
lactose Lactose is a disaccharide sugar synthesized by galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from ' (gen. '), the Latin word for milk, plus the suffix ' ...
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 firstly converted into allolactose by
β-Galactosidase β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. β ...
( lacZ) in bacteria. The DNA binding ability of lac repressor bound with allolactose is inhibited due to
allosteric regulation In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site. The site to which the effector binds is termed the ''allosteric sit ...
, thereby genes coding for proteins involved in lactose uptake and utilization can be expressed.


Function

The ''lac'' repressor (LacI) operates by a
helix-turn-helix Helix-turn-helix is a DNA-binding protein (DBP). The helix-turn-helix (HTH) is a major structural motif capable of binding DNA. Each monomer incorporates two α helices, joined by a short strand of amino acids, that bind to the major groove of ...
motif in its
DNA-binding domain A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence (a recognition sequence) or have a gener ...
, binding base-specifically to the
major groove Major ( commandant in certain jurisdictions) is a military rank of commissioned officer status, with corresponding ranks existing in many military forces throughout the world. When used unhyphenated and in conjunction with no other indicat ...
of the operator region of the ''lac'' operon, with base contacts also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. This bound repressor can reduce transcription of the Lac proteins by occluding the
RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens th ...
binding site or by prompting DNA looping. When lactose is present, allolactose binds to the ''lac'' repressor, causing an
allosteric In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site. The site to which the effector binds is termed the ''allosteric sit ...
change in its shape. In its changed state, the ''lac'' repressor is unable to bind tightly to its cognate operator. Thus, the gene is mostly off in the absence of inducer and mostly on in the presence of inducer, although the degree of gene expression depends on the number of repressors in the cell and on the repressor's DNA-binding affinity. 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
homotetramer A tetrameric protein is a protein with a quaternary structure of four subunits (tetrameric). Homotetramers have four identical subunits (such as glutathione S-transferase), and heterotetramers are complexes of different subunits. A tetramer c ...
. More precisely, the tetramer contains two DNA-binding subunits composed of two monomers each (a dimer of dimers). 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 is a DNA-binding protein (DBP). The helix-turn-helix (HTH) is a major structural motif capable of binding DNA. Each monomer incorporates two α helices, joined by a short strand of amino acids, that bind to the major groove of ...
structural motif In a chain-like biological molecule, such as a protein or nucleic acid, a structural motif is a common three-dimensional structure that appears in a variety of different, evolutionarily unrelated molecules. A structural motif does not have t ...
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 increases the affinity of
RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens th ...
for the promoter sequence such that it cannot enter elongation and remains in
abortive initiation Abortive initiation, also known as abortive transcription, is an early process of genetic transcription in which RNA polymerase binds to a DNA promoter and enters into cycles of synthesis of short mRNA transcripts which are released before the tra ...
. Additionally, because each tetramer contains two DNA-binding subunits, binding of multiple operator sequences by a single tetramer induces DNA looping.


Kinetics of DNA binding and unbinding

LacI finds its target operator DNA surprisingly fast. ''
In vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology a ...
'' the search is 10-100 times faster than the theoretical upper limit for two particles searching for each other via
diffusion Diffusion is the net movement of anything (for example, atoms, ions, molecules, energy) generally from a region of higher concentration to a region of lower concentration. Diffusion is driven by a gradient in Gibbs free energy or chemical ...
in three dimensions (3D). To explain the fast search, it was hypothesized that LacI and other transcriptions factors (TFs) find their binding sites by facilitated diffusion, a combination of free diffusion in 3D and 1D-sliding on the DNA. During sliding the repressor is in contact with the DNA helix, sliding around and tracking its major groove, which speeds up the search process by extending the target length when the TF slides in onto the operator from the side. ''
In vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and p ...
'' single-molecule experiments with ''
E.coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Esche ...
'' cells have now tested and verified the facilitated diffusion model, and shown that the TF scans on average 45 bp during each sliding event, before the TF detaches spontaneously and resumes exploring the genome in 3D. These experiments also suggest that LacI slides over the O1 operator several times before binding, meaning that different DNA sequences can have different probabilities to be recognized at each encounter with the TF. This implies a trade-off between fast search on nonspecific sequences and binding to specific sequences. ''In vivo'' and ''in vitro'' experiments have shown that it is this probability to recognize the operator that changes with DNA sequence, while the time the TF remains in the bound conformation on the operator changes less with sequence. The TF often leaves the sequence it is intended to regulate, but at a strong target site, it almost always make a very short journey before finding the way back again. On the macroscopic scale, this looks like a stable interaction. This binding mechanism explains how DNA binding proteins manage to quickly search through the genome of the cell without getting stuck too long at sequences that resemble the true target. An all-atom
molecular dynamics Molecular dynamics (MD) is a computer simulation method for analyzing the physical movements of atoms and molecules. The atoms and molecules are allowed to interact for a fixed period of time, giving a view of the dynamic "evolution" of th ...
simulation suggests that the transcription factor encounters a barrier of 1 ''kBT'' during sliding and 12 ''kBT'' for dissociation, implying that the repressor will slide over 8 bp on average before dissociating. The ''in vivo'' search model for the lac repressor includes intersegment transfer and hopping as well as crowding by other proteins which make the genome in ''
E.coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Esche ...
'' cells less accessible for the repressor. The existence of hopping, where the protein slips out of the major groove of DNA to land in another nearby groove along the DNA chain, has been proven more directly
in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology a ...
, where the lac repressor has been observed to bypass operators, flip orientation, and rotate with a longer pitch than the 10.5 bp period of DNA while moving along it.


Discovery

The ''lac'' repressor was first
isolated Isolation is the near or complete lack of social contact by an individual. Isolation or isolated may also refer to: Sociology and psychology *Isolation (health care), various measures taken to prevent contagious diseases from being spread **Is ...
by Walter Gilbert and Benno Müller-Hill in 1966. They showed that ''
in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology a ...
'' the protein bound to DNA containing the ''lac'' operon, and it released the DNA when IPTG (an analog of allolactose) was added.


See also

*
Lac operon The ''lactose'' operon (''lac'' operon) is an operon required for the transport and metabolism of lactose in ''E. coli'' and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the ''lac'' operon allow ...


References


External links

* * More information o
the lac repressor molecule
on protein database
Lac Repressor in Proteopedia
{{DEFAULTSORT:Lac Repressor Gene expression Bacterial proteins