Editing Lac repressor

{{Short description|DNA-binding protein}}
{{redirect-distinguish|lacI|lac I|Laci (disambiguation){{!}}Laci}}
[[Image:LacI Dimer Structure Annotated.png|thumb|right|300px|'''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 [[Gene expression|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 firstly converted into [[allolactose]] by [[Beta-galactosidase|β-Galactosidase]] ([[Lac operon|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.

== Function ==
The ''lac'' repressor (LacI) operates by a [[helix-turn-helix]] [[Structural motif|motif]] in its [[DNA-binding domain]], binding base-specifically to the [[major groove]] of the [[Operon|operator region]] of the [[lac operon|''lac'' operon]], with base contacts also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove.<ref>{{cite journal | vauthors = Schumacher MA, Choi KY, Zalkin H, Brennan RG | title = Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices | journal = Science | volume = 266 | issue = 5186 | pages = 763–70 | date = November 1994 | pmid = 7973627 | doi = 10.1126/science.7973627 | bibcode = 1994Sci...266..763S }}</ref> This bound repressor can reduce [[transcription (genetics)|transcription]] of the Lac proteins by occluding the [[RNA polymerase]] binding site or by prompting DNA looping.<ref>{{cite journal | vauthors = Razo-Mejia M, Boedicker J, Jones D, DeLuna A, Kinney J, Phillips R | title = Comparison of the theoretical and real-world evolutionary potential of a genetic circuit | journal = Physical Biology | volume = 1 | issue = 2 | pages = 026005 | date = 2014 | pmid = 24685590 | pmc = 4051709 | doi = 10.1088/1478-3975/11/2/026005| bibcode = 2014PhBio..11b6005R }}</ref> 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.<ref>{{cite journal | vauthors = Razo-Mejia M, Barnes S, Belliveau N, Chure G, Einav T, Lewis M, Phillips R | title = Tuning Transcriptional Regulation through Signaling: A Predictive Theory of Allosteric Induction | journal = Cell Systems | volume = 6 | issue = 4 | pages = 456–469 | date = 2018 | pmid = 29574055 | pmc = 5991102 | doi = 10.1016/j.cels.2018.02.004 }}</ref> [[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 ==
[[File:Annotated Theoretical Model of Bound Tetrameric Lac Repressor.png|thumb|right|300px|'''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 [[Tetrameric protein|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:<ref>{{Cite journal | vauthors = Goodsell DS | doi = 10.2210/rcsb_pdb/mom_2003_3 | title = Lac Repressor | journal = RCSB Protein Data Bank | year = 2003 }}</ref><ref>{{cite journal | vauthors = Lewis M | title = The lac repressor | journal = Comptes Rendus Biologies | volume = 328 | issue = 6 | pages = 521–48 | date = June 2005 | pmid = 15950160 | doi = 10.1016/j.crvi.2005.04.004 | doi-access = free }}</ref><ref name="pmid19269243" />
*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<ref name="pmid19269243">{{cite journal | vauthors = Swint-Kruse L, Matthews KS | title = Allostery in the LacI/GalR family: variations on a theme | journal = Current Opinion in Microbiology | volume = 12 | issue = 2 | pages = 129–37 | date = April 2009 | pmid = 19269243 | pmc = 2688824 | doi = 10.1016/j.mib.2009.01.009 }}</ref>)
*A C-terminal '''tetramerization region''' (which joins four monomers in an alpha-helix bundle)

DNA binding occurs via an N-terminal [[helix-turn-helix]] [[structural motif]] and is targeted to one of several operator DNA sequences (known as O<sub>1</sub>, O<sub>2</sub> and O<sub>3</sub>).  The O<sub>1</sub> 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.<ref name="pmid2182324">{{cite journal | vauthors = Oehler S, Eismann ER, Krämer H, Müller-Hill B | title = The three operators of the lac operon cooperate in repression | journal = The EMBO Journal | volume = 9 | issue = 4 | pages = 973–9 | date = April 1990 | pmid = 2182324 | pmc = 551766 | doi = 10.1002/j.1460-2075.1990.tb08199.x }}</ref>

Each monomer has 360 amino acids, so it has 1440 amino acids in total, and 154,520 Dalton of atomic mass.<ref>{{Cite journal |last=Lewis |first=Mitchell |date=2005-06-01 |title=The lac repressor |url=https://www.sciencedirect.com/science/article/pii/S1631069105000685 |journal=Comptes Rendus Biologies |series=Retour sur l'operon lac |volume=328 |issue=6 |pages=521–548 |doi=10.1016/j.crvi.2005.04.004 |pmid=15950160 |issn=1631-0691|doi-access=free }}</ref>

== Kinetics of DNA binding and unbinding ==

[[File:Binding and unbinding mechanism of LacI.webm|thumb|right|300px|'''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).<ref name="Riggs Bourgeois Cohn 1970 pp. 401–417">{{cite journal | last1=Riggs | first1=Arthur D. | last2=Bourgeois | first2=Suzanne | last3=Cohn | first3=Melvin | title=The lac repressor-operator interaction | journal=Journal of Molecular Biology | publisher=Elsevier BV | volume=53 | issue=3 | year=1970 | issn=0022-2836 | doi=10.1016/0022-2836(70)90074-4 | pages=401–417| pmid=4924006 }}</ref> To explain the fast search, it was hypothesized that LacI and other [[transcription factor]]s (TFs) find their binding sites by facilitated diffusion, a combination of free diffusion in 3D and 1D-sliding on the DNA.<ref name="Berg Winter Von Hippel 1981 pp. 6929–6948">{{cite journal | last1=Berg | first1=Otto G. | last2=Winter | first2=Robert B. | last3=Von Hippel | first3=Peter H. | title=Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory | journal=Biochemistry | publisher=American Chemical Society (ACS) | volume=20 | issue=24 | date=1981-11-01 | issn=0006-2960 | doi=10.1021/bi00527a028 | pages=6929–6948| pmid=7317363 }}</ref> 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 ''[[Escherichia coli|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.<ref name=":0" /> These experiments also suggest that LacI slides over the O<sub>1</sub> 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.<ref name=":0">{{Cite journal|last1=Hammar|first1=Petter|last2=Leroy|first2=Prune|last3=Mahmutovic|first3=Anel|last4=Marklund|first4=Erik G.|last5=Berg|first5=Otto G.|last6=Elf|first6=Johan|date=2012-06-22|title=The lac Repressor Displays Facilitated Diffusion in Living Cells|journal=Science|language=en|volume=336|issue=6088|pages=1595–1598|doi=10.1126/science.1221648|issn=0036-8075|pmid=22723426|bibcode=2012Sci...336.1595H|s2cid=21351861}}</ref> ''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.<ref name="Marklund Mao Yuan Zikrin 2022 pp. 442–445">{{cite journal | last1=Marklund | first1=Emil | last2=Mao | first2=Guanzhong | last3=Yuan | first3=Jinwen | last4=Zikrin | first4=Spartak | last5=Abdurakhmanov | first5=Eldar | last6=Deindl | first6=Sebastian | last7=Elf | first7=Johan | title=Sequence specificity in DNA binding is mainly governed by association | journal=Science | publisher=American Association for the Advancement of Science (AAAS) | volume=375 | issue=6579 | date=2022-01-28 | issn=0036-8075 | doi=10.1126/science.abg7427 | pages=442–445| pmid=35084952 | bibcode=2022Sci...375..442M | s2cid=246360459 | url=http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-466865 }}</ref> 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 [[KT (energy)|''k<sub>B</sub>T'']] during sliding and 12 ''k<sub>B</sub>T'' for dissociation, implying that the repressor will slide over 8 bp on average before dissociating.<ref>{{Cite journal|last1=Marklund|first1=Erik G.|last2=Mahmutovic|first2=Anel|last3=Berg|first3=Otto G.|last4=Hammar|first4=Petter|last5=Spoel|first5=David van der|last6=Fange|first6=David|last7=Elf|first7=Johan|date=2013-12-03|title=Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models|journal=Proceedings of the National Academy of Sciences|language=en|volume=110|issue=49|pages=19796–19801|doi=10.1073/pnas.1307905110|issn=0027-8424|pmid=24222688|pmc=3856812|bibcode=2013PNAS..11019796M|doi-access=free}}</ref> 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 ''[[Escherichia coli|E. coli]]'' cells less accessible for the repressor.<ref>{{Cite journal|last1=Mahmutovic|first1=Anel|last2=Berg|first2=Otto G.|last3=Elf|first3=Johan|date=2015-03-16|title=What matters for lac repressor search in vivo—sliding, hopping, intersegment transfer, crowding on DNA or recognition?|journal=Nucleic Acids Research|language=en|volume=43|issue=7|pages=3454–3464|doi=10.1093/nar/gkv207|issn=1362-4962|pmc=4402528|pmid=25779051}}</ref> 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.<ref name="Marklundvan Oosten2020">{{cite journal|last1=Marklund|first1=Emil|last2=van Oosten|first2=Brad|last3=Mao|first3=Guanzhong|last4=Amselem|first4=Elias|last5=Kipper|first5=Kalle|last6=Sabantsev|first6=Anton|last7=Emmerich|first7=Andrew|last8=Globisch|first8=Daniel|last9=Zheng|first9=Xuan|last10=Lehmann|first10=Laura C.|last11=Berg|first11=Otto G.|last12=Johansson|first12=Magnus|last13=Elf|first13=Johan|last14=Deindl|first14=Sebastian|title=DNA surface exploration and operator bypassing during target search|journal=Nature|volume=583|issue=7818|year=2020|pages=858–861|issn=0028-0836|doi=10.1038/s41586-020-2413-7|pmid=32581356|bibcode=2020Natur.583..858M|s2cid=220049852}}</ref>

==Discovery==
The ''lac'' repressor was first [[protein purification|isolated]] by [[Walter Gilbert]] and [[Benno Müller-Hill]] in 1966.<ref>
{{cite journal | vauthors = Gilbert W, Müller-Hill B | title = Isolation of the lac repressor | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 56 | issue = 6 | pages = 1891–8 | date = December 1966 | pmid = 16591435 | pmc = 220206 | doi = 10.1073/pnas.56.6.1891 | bibcode = 1966PNAS...56.1891G | author-link = Walter Gilbert | doi-access = free }}
</ref> They showed that ''[[in vitro]]'' the protein bound to DNA containing the ''lac'' operon, and it released the DNA when [[IPTG]] (an [[analog (chemistry)|analog]] of allolactose) was added. 

== See also ==
*[[Lac operon]]

== References ==
{{reflist|2}}

== External links ==
* {{MeshName|Lac Repressor}}
* More information on [http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb39_1.html the lac repressor molecule] {{Webarchive|url=https://web.archive.org/web/20100528025339/http://www.pdb.org/pdb/static.do?p=education_discussion%2Fmolecule_of_the_month%2Fpdb39_1.html |date=2010-05-28 }} on protein database
* [http://proteopedia.org/wiki/index.php/Lac_repressor Lac Repressor in Proteopedia].

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[[Category:Gene expression]]
[[Category:Bacterial proteins]]
[[Category:Lactose]]