The ''lac'' repressor (LacI) is a DNA-binding protein that inhibits the
expression
Expression may refer to:
Linguistics
* Expression (linguistics), a word, phrase, or sentence
* Fixed expression, a form of words with a specific meaning
* Idiom, a type of fixed expression
* Metaphorical expression, a particular word, phrase, o ...
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 ba ...
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 cell ...
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
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 the occasional transglycosylation ...
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 catalyst, catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosi ...
(
lacZ
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 allows ...
) 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 site ...
, 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 D ...
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 genera ...
, 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 indicators ...
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
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
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 the ...
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 site ...
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
Isopropyl β--1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers Transcription (genetics), transcription of the lac operon, ''lac'' operon, and it ...
(IPTG) is a commonly used allolactose mimic which can be used to induce transcription of genes being regulated by ''lac'' repressor.
Structure
![Annotated Theoretical Model of Bound Tetrameric Lac Repressor](https://upload.wikimedia.org/wikipedia/commons/9/93/Annotated_Theoretical_Model_of_Bound_Tetrameric_Lac_Repressor.png)
Structurally, the ''lac'' repressor protein is a
homotetramer
A tetrameric protein is a protein with a protein quaternary structure, quaternary structure of four subunits (tetrameric). Homotetramers have four identical Protein subunit, subunits (such as glutathione S-transferase), and heterotetramers are M ...
. 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 D ...
structural motif
In a polymer, chain-like biological molecule, such as a protein or nucleic acid, a structural motif is a common Biomolecular structure#Tertiary structure, three-dimensional structure that appears in a variety of different, evolutionarily unrel ...
and is targeted to one of several operator DNA sequences (known as O
1, O
2 and O
3). The O
1 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 the ...
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 an ...
'' 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 p ...
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 ...
'' 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 O
1 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 the ...
simulation suggests that the transcription factor encounters a barrier of 1
''kBT'' during sliding and 12 ''k
BT'' 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 an ...
, 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
Walter Gilbert (born March 21, 1932) is an American biochemist, physicist, molecular biology pioneer, and Nobel laureate.
Education and early life
Walter Gilbert was born in Boston, Massachusetts, on March 21, 1932, the son of Emma (Cohen), a ...
and
Benno Müller-Hill
Benno Müller-Hill (5 February 1933 – 11 August 2018) was a German biologist and author. Together with Walter Gilbert, Müller-Hill purified the lac repressor
The ''lac'' repressor (LacI) is a DNA-binding protein that inhibits the expression of ...
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 an ...
'' the protein bound to DNA containing the ''lac'' operon, and it released the DNA when
IPTG (an
analog
Analog or analogue may refer to:
Computing and electronics
* Analog signal, in which information is encoded in a continuous variable
** Analog device, an apparatus that operates on analog signals
*** Analog electronics, circuits which use 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 allows ...
References
External links
*
* More information o
the lac repressor moleculeon protein database
Lac Repressor in Proteopedia
{{DEFAULTSORT:Lac Repressor
Gene expression
Bacterial proteins