Lac repressor

Last updated
Annotated crystal structure of dimeric LacI. Two monomers (of four total) co-operate to bind each DNA operator sequence. Monomers (red and blue) contain DNA binding and core domains (labeled) which are connected by a linker (labeled). The C-terminal tetramerization helix is not shown. The repressor is shown in complex with operator DNA (gold) and ONPF (green), an anti-inducer ligand (i.e. a stabilizer of DNA binding) LacI Dimer Structure Annotated.png
Annotated crystal structure of dimeric LacI. Two monomers (of four total) co-operate to bind each DNA operator sequence. Monomers (red and blue) contain DNA binding and core domains (labeled) which are connected by a linker (labeled). The C-terminal tetramerization helix is not shown. The repressor is shown in complex with operator DNA (gold) and ONPF (green), an anti-inducer ligand (i.e. a stabilizer of DNA binding)

The lac repressor (LacI) is a DNA-binding protein that inhibits the expression of genes 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 firstly converted into allolactose by β-Galactosidase (lacZ) in bacteria. The DNA binding ability of lac repressor bound with allolactose is inhibited due to allosteric regulation, thereby genes coding for proteins involved in lactose uptake and utilization can be expressed.

Contents

Function

The lac repressor (LacI) operates by a helix-turn-helix motif in its DNA-binding domain, binding base-specifically to the major groove 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. [1] This bound repressor can reduce transcription of the Lac proteins by occluding the RNA polymerase binding site or by prompting DNA looping. [2] 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. 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. [3] 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

Tetrameric LacI binds two operator sequences and induces DNA looping. Two dimeric LacI functional subunits (red+blue and green+orange) each bind a DNA operator sequence (labeled). These two functional subunits are coupled at the tetramerization region (labeled); thus, tetrameric LacI binds two operator sequences. This allows tetrameric LacI to induce DNA looping. Annotated Theoretical Model of Bound Tetrameric Lac Repressor.png
Tetrameric LacI binds two operator sequences and induces DNA looping. Two dimeric LacI functional subunits (red+blue and green+orange) each bind a DNA operator sequence (labeled). These two functional subunits are coupled at the tetramerization region (labeled); thus, tetrameric LacI binds two operator sequences. This allows tetrameric LacI to induce DNA looping.

Structurally, the lac repressor protein is a homotetramer. 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: [4] [5] [6]

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 increases the affinity of RNA polymerase for the promoter sequence such that it cannot enter elongation and remains in abortive initiation. Additionally, because each tetramer contains two DNA-binding subunits, binding of multiple operator sequences by a single tetramer induces DNA looping. [7]

Each monomer has 360 amino acids, so it has 1440 amino acids in total, and 154,520 Dalton of atomic mass. [8]

Kinetics of DNA binding and unbinding

Animation of binding and unbinding mechanism of a LacI dimer and its target DNA site.

LacI finds its target operator DNA surprisingly fast. In vitro the search is 10-100 times faster than the theoretical upper limit for two particles searching for each other via diffusion in three dimensions (3D). [9] To explain the fast search, it was hypothesized that LacI and other transcription factors (TFs) find their binding sites by facilitated diffusion, a combination of free diffusion in 3D and 1D-sliding on the DNA. [10] 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 single-molecule experiments with E.coli 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. [11] 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. [11] 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. [12] 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 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. [13] 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 cells less accessible for the repressor. [14] 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, 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. [15]

Discovery

The lac repressor was first isolated by Walter Gilbert and Benno Müller-Hill in 1966. [16] They showed that in vitro the protein bound to DNA containing the lac operon, and it released the DNA when IPTG (an analog of allolactose) was added.

See also

Related Research Articles

<span class="mw-page-title-main">Facilitated diffusion</span> Biological process

Facilitated diffusion is the process of spontaneous passive transport of molecules or ions across a biological membrane via specific transmembrane integral proteins. Being passive, facilitated transport does not directly require chemical energy from ATP hydrolysis in the transport step itself; rather, molecules and ions move down their concentration gradient according to the principles of diffusion.

In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lactose 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 enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of β-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

A transcriptional activator is a protein that increases transcription of a gene or set of genes. Activators are considered to have positive control over gene expression, as they function to promote gene transcription and, in some cases, are required for the transcription of genes to occur. Most activators are DNA-binding proteins that bind to enhancers or promoter-proximal elements. The DNA site bound by the activator is referred to as an "activator-binding site". The part of the activator that makes protein–protein interactions with the general transcription machinery is referred to as an "activating region" or "activation domain".

<span class="mw-page-title-main">DNA-binding protein</span> Proteins that bind with DNA, such as transcription factors, polymerases, nucleases and histones

DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that identify a base pair.

<span class="mw-page-title-main">Helix-turn-helix</span> Structural motif capable of binding DNA

Helix-turn-helix is a DNA-binding domain (DBD). 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 DNA. The HTH motif occurs in many proteins that regulate gene expression. It should not be confused with the helix–loop–helix motif.

Isopropyl β-<small>D</small>-1-thiogalactopyranoside Chemical compound

Isopropyl β-d-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce protein expression where the gene is under the control of the lac operator.

<span class="mw-page-title-main">Repressor</span> Sort of RNA-binding protein in molecular genetics

In molecular genetics, a repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to the mRNA and prevents translation of the mRNA into protein. This blocking or reducing of expression is called repression.

<span class="mw-page-title-main">Leucine zipper</span> DNA-binding structural motif

A leucine zipper is a common three-dimensional structural motif in proteins. They were first described by Landschulz and collaborators in 1988 when they found that an enhancer binding protein had a very characteristic 30-amino acid segment and the display of these amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The polypeptide segments containing these periodic arrays of leucine residues were proposed to exist in an alpha-helical conformation and the leucine side chains from one alpha helix interdigitate with those from the alpha helix of a second polypeptide, facilitating dimerization.

<span class="mw-page-title-main">Catabolite activator protein</span> Trans-acting transcriptional activator

Catabolite activator protein is a trans-acting transcriptional activator that exists as a homodimer in solution. Each subunit of CAP is composed of a ligand-binding domain at the N-terminus and a DNA-binding domain at the C-terminus. Two cAMP molecules bind dimeric CAP with negative cooperativity. Cyclic AMP functions as an allosteric effector by increasing CAP's affinity for DNA. CAP binds a DNA region upstream from the DNA binding site of RNA Polymerase. CAP activates transcription through protein-protein interactions with the α-subunit of RNA Polymerase. This protein-protein interaction is responsible for (i) catalyzing the formation of the RNAP-promoter closed complex; and (ii) isomerization of the RNAP-promoter complex to the open conformation. CAP's interaction with RNA polymerase causes bending of the DNA near the transcription start site, thus effectively catalyzing the transcription initiation process. CAP's name is derived from its ability to affect transcription of genes involved in many catabolic pathways. For example, when the amount of glucose transported into the cell is low, a cascade of events results in the increase of cytosolic cAMP levels. This increase in cAMP levels is sensed by CAP, which goes on to activate the transcription of many other catabolic genes.

<span class="mw-page-title-main">Silencer (genetics)</span> Type of DNA sequence

In genetics, a silencer is a DNA sequence capable of binding transcription regulation factors, called repressors. DNA contains genes and provides the template to produce messenger RNA (mRNA). That mRNA is then translated into proteins. When a repressor protein binds to the silencer region of DNA, RNA polymerase is prevented from transcribing the DNA sequence into RNA. With transcription blocked, the translation of RNA into proteins is impossible. Thus, silencers prevent genes from being expressed as proteins.

<span class="mw-page-title-main">Regulator gene</span> Gene involved in controlling expression of other genes

In genetics, a regulator gene, regulator, or regulatory gene is a gene involved in controlling the expression of one or more other genes. Regulatory sequences, which encode regulatory genes, are often at the five prime end (5') to the start site of transcription of the gene they regulate. In addition, these sequences can also be found at the three prime end (3') to the transcription start site. In both cases, whether the regulatory sequence occurs before (5') or after (3') the gene it regulates, the sequence is often many kilobases away from the transcription start site. A regulator gene may encode a protein, or it may work at the level of RNA, as in the case of genes encoding microRNAs. An example of a regulator gene is a gene that codes for a repressor protein that inhibits the activity of an operator.

In molecular biology, an inducer is a molecule that regulates gene expression. An inducer functions in two ways; namely:

<span class="mw-page-title-main">TetR</span>

Tet Repressor proteins are proteins playing an important role in conferring antibiotic resistance to large categories of bacterial species.

<i>trp</i> operon Operon that codes for the components for production of tryptophan

The trp operon is a group of genes that are transcribed together, encoding the enzymes that produce the amino acid tryptophan in bacteria. The trp operon was first characterized in Escherichia coli, and it has since been discovered in many other bacteria. The operon is regulated so that, when tryptophan is present in the environment, the genes for tryptophan synthesis are repressed.

The L-arabinose operon, also called the ara or araBAD operon, is an operon required for the breakdown of the five-carbon sugar L-arabinose in Escherichia coli. The L-arabinose operon contains three structural genes: araB, araA, araD, which encode for three metabolic enzymes that are required for the metabolism of L-arabinose. AraB (ribulokinase), AraA, and AraD produced by these genes catalyse conversion of L-arabinose to an intermediate of the pentose phosphate pathway, D-xylulose-5-phosphate.

The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. Repression of gene expression for this operon works via binding of repressor molecules to two operators. These repressors dimerize, creating a loop in the DNA. The loop as well as hindrance from the external operator prevent RNA polymerase from binding to the promoter, and thus prevent transcription. Additionally, since the metabolism of galactose in the cell is involved in both anabolic and catabolic pathways, a novel regulatory system using two promoters for differential repression has been identified and characterized within the context of the gal operon.

<span class="mw-page-title-main">Catabolite Control Protein A</span>

Catabolite Control Protein A (CcpA) is a master regulator of carbon metabolism in gram-positive bacteria. It is a member of the LacI/GalR transcription regulator family. In contrast to most LacI/GalR proteins, CcpA is allosterically regulated principally by a protein-protein interaction, rather than a protein-small molecule interaction. CcpA interacts with the phosphorylated form of Hpr and Crh, which is formed when high concentrations of glucose or fructose-1,6-bisphosphate are present in the cell. Interaction of Hpr or Crh modulates the DNA sequence specificity of CcpA, allowing it to bind operator DNA to modulate transcription. Small molecules glucose-6-phosphate and fructose-1,6-bisphosphate are also known allosteric effectors, fine-tuning CcpA function.

The nik operon is an operon required for uptake of nickel ions into the cell. It is present in many bacteria, but has been extensively studied in Helicobacter pylori. Nickel is an essential nutrient for many microorganisms, where it participates in a variety of cellular processes. However, excessive levels of nickel ions in cell can be fatal to the cell. Nickel ion concentration in the cell is regulated through the nik operon.

<span class="mw-page-title-main">Kathleen Matthews (biochemist)</span> American Biochemist

Kathleen Matthews is an American biochemist specializing in DNA/protein interactions, specifically related to the lac repressor. She is the Stewart Memorial Professor Emerita of BioSciences at Rice University.

References

  1. ↑ Schumacher MA, Choi KY, Zalkin H, Brennan RG (November 1994). "Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices". Science. 266 (5186): 763–70. Bibcode:1994Sci...266..763S. doi:10.1126/science.7973627. PMID   7973627.
  2. ↑ Razo-Mejia M, Boedicker J, Jones D, DeLuna A, Kinney J, Phillips R (2014). "Comparison of the theoretical and real-world evolutionary potential of a genetic circuit". Physical Biology. 1 (2): 026005. Bibcode:2014PhBio..11b6005R. doi:10.1088/1478-3975/11/2/026005. PMC   4051709 . PMID   24685590.
  3. ↑ Razo-Mejia M, Barnes S, Belliveau N, Chure G, Einav T, Lewis M, Phillips R (2018). "Tuning Transcriptional Regulation through Signaling: A Predictive Theory of Allosteric Induction". Cell Systems. 6 (4): 456–469. doi:10.1016/j.cels.2018.02.004. PMC   5991102 . PMID   29574055.
  4. ↑ Goodsell DS (2003). "Lac Repressor". RCSB Protein Data Bank. doi:10.2210/rcsb_pdb/mom_2003_3.
  5. ↑ Lewis M (June 2005). "The lac repressor". Comptes Rendus Biologies. 328 (6): 521–48. doi:10.1016/j.crvi.2005.04.004. PMID   15950160.
  6. 1 2 Swint-Kruse L, Matthews KS (April 2009). "Allostery in the LacI/GalR family: variations on a theme". Current Opinion in Microbiology. 12 (2): 129–37. doi:10.1016/j.mib.2009.01.009. PMC   2688824 . PMID   19269243.
  7. ↑ Oehler S, Eismann ER, Krämer H, Müller-Hill B (April 1990). "The three operators of the lac operon cooperate in repression". The EMBO Journal. 9 (4): 973–9. doi:10.1002/j.1460-2075.1990.tb08199.x. PMC   551766 . PMID   2182324.
  8. ↑ Lewis, Mitchell (2005-06-01). "The lac repressor". Comptes Rendus Biologies. Retour sur l'operon lac. 328 (6): 521–548. doi:10.1016/j.crvi.2005.04.004. ISSN   1631-0691.
  9. ↑ Riggs, Arthur D.; Bourgeois, Suzanne; Cohn, Melvin (1970). "The lac represser-operator interaction". Journal of Molecular Biology. 53 (3). Elsevier BV: 401–417. doi:10.1016/0022-2836(70)90074-4. ISSN   0022-2836. PMID   4924006.
  10. ↑ Berg, Otto G.; Winter, Robert B.; Von Hippel, Peter H. (1981-11-01). "Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory". Biochemistry. 20 (24). American Chemical Society (ACS): 6929–6948. doi:10.1021/bi00527a028. ISSN   0006-2960. PMID   7317363.
  11. 1 2 Hammar, Petter; Leroy, Prune; Mahmutovic, Anel; Marklund, Erik G.; Berg, Otto G.; Elf, Johan (2012-06-22). "The lac Repressor Displays Facilitated Diffusion in Living Cells". Science. 336 (6088): 1595–1598. Bibcode:2012Sci...336.1595H. doi:10.1126/science.1221648. ISSN   0036-8075. PMID   22723426. S2CID   21351861.
  12. ↑ Marklund, Emil; Mao, Guanzhong; Yuan, Jinwen; Zikrin, Spartak; Abdurakhmanov, Eldar; Deindl, Sebastian; Elf, Johan (2022-01-28). "Sequence specificity in DNA binding is mainly governed by association". Science. 375 (6579). American Association for the Advancement of Science (AAAS): 442–445. doi:10.1126/science.abg7427. ISSN   0036-8075. PMID   35084952. S2CID   246360459.
  13. ↑ Marklund, Erik G.; Mahmutovic, Anel; Berg, Otto G.; Hammar, Petter; Spoel, David van der; Fange, David; Elf, Johan (2013-12-03). "Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models". Proceedings of the National Academy of Sciences. 110 (49): 19796–19801. Bibcode:2013PNAS..11019796M. doi: 10.1073/pnas.1307905110 . ISSN   0027-8424. PMC   3856812 . PMID   24222688.
  14. ↑ Mahmutovic, Anel; Berg, Otto G.; Elf, Johan (2015-03-16). "What matters for lac repressor search in vivo—sliding, hopping, intersegment transfer, crowding on DNA or recognition?". Nucleic Acids Research. 43 (7): 3454–3464. doi:10.1093/nar/gkv207. ISSN   1362-4962. PMC   4402528 . PMID   25779051.
  15. ↑ Marklund, Emil; van Oosten, Brad; Mao, Guanzhong; Amselem, Elias; Kipper, Kalle; Sabantsev, Anton; Emmerich, Andrew; Globisch, Daniel; Zheng, Xuan; Lehmann, Laura C.; Berg, Otto G.; Johansson, Magnus; Elf, Johan; Deindl, Sebastian (2020). "DNA surface exploration and operator bypassing during target search". Nature. 583 (7818): 858–861. Bibcode:2020Natur.583..858M. doi:10.1038/s41586-020-2413-7. ISSN   0028-0836. PMID   32581356. S2CID   220049852.
  16. ↑ Gilbert W, Müller-Hill B (December 1966). "Isolation of the lac repressor". Proceedings of the National Academy of Sciences of the United States of America. 56 (6): 1891–8. Bibcode:1966PNAS...56.1891G. doi: 10.1073/pnas.56.6.1891 . PMC   220206 . PMID   16591435.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.