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Lac repressor

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Morph of the lac repressor complexed with DNA showing the differences between non-specific binding (straight DNA) vs. specific recognition of the operator sequence (kinked DNA). Whether the binding kinks the DNA, or simply stabilizes a pre-existing kink, is unknown. Details Below.

Contents

What is the lac repressor?

Repressors are proteins that inhibit the expression of genes; that is, they inhibit the transcription of messenger RNA from their target genes. Each repressor targets a specific co-regulated group of genes by recognizing a specific sequence of DNA, called the operator in bacteria. Repressor proteins are coded for by regulatory genes.

The lactose ("lac") repressor controls the expression of bacterial enzymes involved in the metabolism of of the sugar lactose. When the lac repressor binds lactose, it changes to an inactive conformation that cannot repress the production of these enzymes. Thus, the enzymes needed to use lactose are made only when lactose is available. The lac repressor, and the group of genes it controls, which is called an operon, were the first such gene regulatory system to be discovered. The operon was described in 1960[1] by François Jacob et al., who also correctly proposed the general mechanism of regulation by the lac repressor. The 1965 Nobel Prize in Physiology or Medicine was awarded to François Jacob, André Lwoff, and Jacques Monod "for their discoveries concerning genetic control of enzyme and virus synthesis".

For a general introduction to the lac repressor, please see David Goodsell's Introduction to the lac repressor in his series Molecule of the Month, and the article in Wikipedia on the lac repressor. Mitchell Lewis published a detailed review in 2005[2]. See also Transcription and RNA Processing.

Structure of the lac repressor

The lac repressor protein ( showing chain A in 1lbg, resolution 4.8 Å), starting at the N-terminus, begins with a DNA-binding "headpiece", followed by a hinge region, then an N-terminal ligand-binding subdomain and a C-terminal ligand binding subdomain, a linker, and a C-terminal tetramerization helix[3]. (.) In the absence of DNA, the hinge region does not form the alpha helix shown here.

As can be seen when the chain is

N               C

each of the ligand-binding subdomains is made up of two discontinuous segments.

The lac repressor forms . Dimerization buries 2,200 Å2 of surface, including a ,

Hydrophobic, Polar

forming a hydrophobic core (shown with 1lbi, resolution 2.7 Å, lacking the DNA-binding domain due to disorder).

 Image:Consurf_key_small.gif

The most highly is the surface that contacts DNA[4]. (Only alpha carbon atoms are shown here, without sidechains, because sidechains were not resolved in the 4.8 Å 1lbg model.) The dimerization surfaces are the of the ligand-binding domains[5]. (This scene shows sidechains, using the 2.7 Å model in 1lbi, which lacks the DNA-binding domain due to disorder.)

The C-terminal tetramerization helices tether two dimers, and thus the functional form of with two DNA-binding sites.

DNA Binding: A Kink In The Operator

Non-Specific Binding

Lac repressor binds to DNA non-specifically ( derived [6] from 1osl, 20 NMR models), enabling it to slide rapidly along the DNA double helix until it encounters the lac operator sequence ("facilitated diffusion"[7]). The DNA-binding domain employs a helix-turn-helix motif (Alpha Helices, Turns). During non-specific binding, the hinge region is disordered (indicated by the range of positions of the 20 models). The DNA double helix is depicted as straight in the model shown here (see methods), but in actuality, straightness likely varies with sequence (see below). The protein model shown at right (1osl) has two copies of the DNA-binding domain and hinge region ( to distinguish the chain B hinge). these 20 NMR models simulates thermal motion of the disordered hinge regions.


Specific Binding

Upon recognizing the specific operator sequence, the non-specific binding converts to (derived[6] from 1l1m, 20 NMR models). During this conversion, the hinge region changes from disordered loops to Alpha Helices (), which bind to the minor groove of the DNA. As explained below, this binding stabilizes a kinked ("bent") DNA double helix conformation. What percentage of time this DNA sequence spends in a kinked state, in the absence of bound lac repressor protein, is not known, but it may be a significant percentage (see next section below). these 20 NMR models can be compared with the animation of the non-specific binding. See Lac repressor morph methods.


DNA Recognition

Proteins typically recognize specific DNA sequences in the major groove by direct readout, that is, by forming sequence-specific hydrogen bonds with the edges of the DNA bases exposed in the major groove[8].

DNA sequence recognition in the minor groove, often accompanied by kinking or bending of the DNA, is more complex. Direct readout is less important, since, unlike in the major groove, the four bases do not present unique hydrogen-bonding surfaces in the minor groove[8]. Recognition of the shape of the DNA seems more important[9][10]. In many cases, cationic arginines are believed to be attracted to a region of the minor groove with high aninoic charge density resulting from narrowing of the groove[10]. In these cases, the protein appears to recognize the shape of the DNA minor groove (indirect readout)[10].

In the lac repressor complex with specific DNA, a pair of arginines (Arg51 in each chain) is close to the minor groove, but points away from the groove (). Hence the binding of arginines to narrow minor grooves does not appear to be involved in specific DNA recognition by the lac repressor.

DNA Kinks

Strictly speaking, bends in DNA are distinguished from kinks. DNA is said to be kinked when the stacking contact between two adjacent base pairs is disrupted[8]. The DNA on either side of a kink may be straight or bent. A : a single CpG base pair is partially separated from the adjacent CpG base pair. . Pyrimidine-purine base pairs have the weakest stacking interactions, and are most susceptible to kinking[8]. In the complex of lac repressor with specific DNA, are partially interchalated between the separated CpG base pairs, which helps to stabilize the kink. It may often be the case that sequence-dependent kinks and bends are present in DNA prior to the binding of protein[8]. DNA structure is dynamic. For example, recently Hoogsteen base pairing was observed to occur transiently in equilibrium with Watson-Crick base pairing[11] (See News & Views[12]). Also, the binding of p53 to some but not all DNA sequences stabilizes Hoogsteen (rather than Watson-Crick) base pairing[13]. Thus, the "bending" (actually kinking) depicted in the morph on this page may give the wrong impression: lac repressor binding may simply stabilize a kink (or transient kink) that pre-existed in the cognate DNA sequence.

DNA Bends

Strictly speaking, bends in DNA are distinguished from kinks. Bending means a curvature distributed over several adjacent base pairs[8], whereas a kink (see previous section above) is a disruption in stacking limited to a single pair of adjacent base pairs. An is found in the DNA complexed to HPV E2 protein (thanks to [8] for this example, 1jj2).

Morph of Conversion

The can be seen more easily when they are animated smoothly by morphing. (The methods used to create this morph are given in Lac repressor morph methods.)

  • Note the kinking of the DNA, with the widening of the central minor groove on the convex aspect. Whether the cognate DNA sequence is (perhaps transiently) kinked prior to lac repressor binding is unknown (see above).
  • Also note the conversion of the disulfide-bonded hinge region loops to alpha helices. (The displayed secondary structure is calculated for each model in the morph interpolation.)

The specific recognition of the lac operator sequence in the DNA occurs largely though hydrogen bonds. is illustrated in this rendering of the morph. Shown are hydrogen bonds involving Arg22.N-eta2 and Tyr18.OH interacting with DNA base oxygens in the major groove, and Ala53.O interacting with a DNA base nitrogen in the minor groove. (Not all of the relevant hydrogen bonds are shown; see Methods.)


Animation for Powerpoint® Slides

Here is an animated multi-gif true movie of the above morph, ready to insert into a Powerpoint®[14] slide. If the image below is not moving, reload this page (it stops after 50 cycles).

  • In Windows, simply drag the movie and drop it into the Powerpoint slide. You can then resize it and position it. The movie should play when you change the View to Slide Show ("project") the slide.
  • In Mac OSX, Ctrl-Click on the movie, then Save Image. In Mac Powerpoint, at the desired slide, use the Insert menu (at the top) and select Movie ..., then insert the saved .gif movie file. After inserting the movie, make sure the Toolbox is showing (controlled with an icon-button at the top of the window). Now you can resize and reposition the movie. Click in the movie in the slide to select it. Now, in the Toolbox/Formatting Palette, under Movie, check Loop Until Stopped. Now the movie should play when you change the View to Slide Show ("project") the slide.

Challenge Your Understanding

Here are some questions to challenge your understanding.

  1. Why does the lac repressor bind to DNA non-specifically?
  2. When the lac repressor binds non-specifically to DNA, what part of the DNA double helix does it bind to?
  3. Does DNA have a net charge, and if so, is it negative or positive in aqueous solution at pH 7?
  4. What kinds of chemical bonds are likely to be involved in non-specific binding of the repressor protein to DNA?
  5. Does specific binding of lac repressor to DNA disrupt any of the Watson-Crick hydrogen bonds between the base pairs in the DNA strands?
  6. How do proteins such as the lac repressor recognize specific nucleotide sequences in a DNA double helix?
  7. What kinds of chemical bonds are involved in specific binding of the repressor protein to DNA?
  8. Does the lac repressor recognize specific bases in the major or minor grooves of the DNA?
  9. When it recognizes its specific nucleotide sequence, how does the lac repressor stabilize a kink in the DNA double helix?

Answers are available on request to Image:Contact-email.png. If you would like us to make the answers publically available within Proteopedia, please let us know. When contacting us, please give your full name, your position, institution or school, and location.

Content Attribution & Acknowledgement

The morphs displayed here were originally prepared by Eric Martz in 2004 for the page Lac Repressor Binding to DNA, within ProteinExplorer.Org.

Eric Martz thanks Remo Rohs for his kind and expert advice concerning the 2010-2011 updates to this article.

See Also


3D structures of Lac repressor

Updated on 21-July-2013

3edc – EcLAC + hexanediol - Escherichia coli
2pe5 – EcLAC residues 2-331 (mutant) + effector
1lbh - EcLAC + effector
2p9h - EcLAC residues 62-330 + effector
2paf - EcLAC residues 62-330 + anti-inducer
1lbi – EcLAC
1jye, 1jyf - EcLAC (mutant)
1lqc - EcLAC headpiece – NMR
1tlf - EcLAC residues 19-319
2r2v – LAC coiled-coil - yeast

Lac repressor complex with DNA

2kei, 1l1m – EcLAC DNA-binding domain (mutant) + O1 operator –NMR
2kej - EcLAC DNA-binding domain (mutant) + O2 operator – NMR
2kek - EcLAC DNA-binding domain (mutant) + O3 operator – NMR
2bjc - EcLAC DNA-binding domain (mutant) + GAL operator – NMR
1osl - EcLAC DNA-binding domain (mutant) + DNA – NMR
1cjg, 1lcc, 1lcd - EcLAC headpiece + DNA – NMR
1jwl - EcLAC + O1 operator + effector
1lbg - EcLAC + DNA + inducer
1efa - EcLAC residues 1-333 (mutant) + DNA


References & Notes

  1. L'opéron: groupe de gènes à expression coordonée par un opérateur. [Operon: a group of genes with the expression coordinated by an operator.] C R Hebd Seances Acad Sci., 250:1727-9, 1960. PubMed 14406329
  2. The lac repressor. Lewis, M. C R Biol. 328:521-48, 2005. PubMed 15950160
  3. This domain coloring scheme is adapted from Fig. 6 in the review by Lewis (C. R. Biol. 328:521, 2005). Domains are 1-45, 46-62, (63-162,291-320), (163-290,321-332), 330-339, and 340-357.
  4. Conservation results for 1lbg are from the precalculated ConSurf Database, using 103 sequences from Swiss-Prot with an average pairwise distance of 2.4.
  5. Conservation results for 1lbi are from the ConSurf Server, using 100 sequences from Uniprot with an average pairwise distance of 1.3.
  6. 6.0 6.1 For these scenes, the 20-model PDB files for 1osl and 1l1m were reduced in size, to avoid exceeding the java memory available to the Jmol applet. All atoms except amino acid alpha carbons and DNA phosphorus atoms were removed using the free program alphac.exe from PDBTools. Secondary structure HELIX records from the original PDB file header were retained. The results are Image:1osl ca.pdb and Image:1l1m ca.pdb.
  7. Hammar P, Leroy P, Mahmutovic A, Marklund EG, Berg OG, Elf J. The lac repressor displays facilitated diffusion in living cells. Science. 2012 Jun 22;336(6088):1595-8. PMID:22723426 doi:10.1126/science.1221648
  8. 8.0 8.1 8.2 8.3 8.4 8.5 8.6 Rohs R, Jin X, West SM, Joshi R, Honig B, Mann RS. Origins of specificity in protein-DNA recognition. Annu Rev Biochem. 2010;79:233-69. PMID:20334529 doi:10.1146/annurev-biochem-060408-091030
  9. Joshi R, Passner JM, Rohs R, Jain R, Sosinsky A, Crickmore MA, Jacob V, Aggarwal AK, Honig B, Mann RS. Functional specificity of a Hox protein mediated by the recognition of minor groove structure. Cell. 2007 Nov 2;131(3):530-43. PMID:17981120 doi:10.1016/j.cell.2007.09.024
  10. 10.0 10.1 10.2 Rohs R, West SM, Sosinsky A, Liu P, Mann RS, Honig B. The role of DNA shape in protein-DNA recognition. Nature. 2009 Oct 29;461(7268):1248-53. PMID:19865164 doi:10.1038/nature08473
  11. Nikolova EN, Kim E, Wise AA, O'Brien PJ, Andricioaei I, Al-Hashimi HM. Transient Hoogsteen base pairs in canonical duplex DNA. Nature. 2011 Feb 24;470(7335):498-502. Epub 2011 Jan 26. PMID:21270796 doi:10.1038/nature09775
  12. Honig B, Rohs R. Biophysics: Flipping Watson and Crick. Nature. 2011 Feb 24;470(7335):472-3. PMID:21350476 doi:10.1038/470472a
  13. Kitayner M, Rozenberg H, Rohs R, Suad O, Rabinovich D, Honig B, Shakked Z. Diversity in DNA recognition by p53 revealed by crystal structures with Hoogsteen base pairs. Nat Struct Mol Biol. 2010 Apr;17(4):423-9. Epub 2010 Apr 4. PMID:20364130 doi:10.1038/nsmb.1800
  14. Powerpoint is a registered trademark for a software package licensed by Microsoft Corp..

Proteopedia Page Contributors and Editors (what is this?)

Eric Martz, Michal Harel, David Canner, Alexander Berchansky, Eran Hodis

DOI: http://dx.doi.org/10.14576/324680.1823276 (?)
Citation: Martz E, Hodis E, Canner D, Harel M, Berchansky A, 2013, "Lac repressor", Proteopedia, DOI: http://dx.doi.org/10.14576/324680.1823276
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