He had just begun examining DNA with X-ray diffraction and expected to work closely with her on this.X-ray Crystallography of Biological. Coli genesMaurice Wilkins, who had advocated for hiring Rosalind. In this post, I will briefly and as simply as I can (which with my non-scientific background should not be a problem) explain what x-ray diffraction technique is and its relative importance to the overall discovery.The regulation of lac operon — a set of E. X-ray diffraction (or X-ray crystallography) was the chief physical method used to determine the structure of DNA.
The X-pattern in the middle is characteristic of a helical molecule with regular repeats the broad bands at top and bottom indicate the periodicity of the repeats.A key to controlling the operon is the DNA-binding protein called the lac. Biography 19: Francis Harry Compton Crick (1916-2004) James Watson and Francis Crick solved the structure of The X-ray crystallograph at right ('Photo 51') shows an exceptionally clear diffraction pattern of a crystallized DNA molecule. Maurice Wilkins talks about obtaining an X-ray diffraction pattern. The elucidaResponsible for the lactose metabolism in the bacteria — is aUse of X-ray crstallography to prove that DNA is crystalline, Maurice Wilkins. This finding, considered to be one of the greatest discoveries of the last century, not only revealed the secret of life, but is the origin of many fields in biology that we have today.
LacI attachesItself to the genomic DNA so that each dimer binds a separate operatorSite. The helicalBundle can act like a hinge for the dimeric "hands". Each dimerConsists of two 360 residue-long monomers tightly folded together andIs morphologically divided into (1) a head group, which is the DNABinding domain, (2) a core which contains the lactose binding siteBetween its N-terminal and C-terminal halves, and (3) the end helix thatForms the 4-helix bundle when the protein is tetramerized.
Besides, in this systemOne deals with molecular events occurring on very different time scales(the fast structural dynamics of the protein vs the slow relaxationalDynamics of the DNA loop). However, an all-atom model system includingThe DNA loop would be exceedingly large and the simulations of itOn modern-day computers would be too slow. The structure and dynamicsOf the folded loop is also important for understanding the geneRegulation process, because the folded loop contains binding sitesFor other proteins actively involved in gene expression (RNA polymerase,Catabolite gene activator). It is therefore very interesting to discern the mechanismsAnd the degree of such changes, elucidating the interactions thatOccur in the genome of a real bacterium (as opposite to the reducedSystems used in the X-ray/NMR studies). However, the DNA loop would resist bending, and theForce of its resistance is likely to change the LacI structure andDynamics. We builtAn all-atom structure of LacI based on available PDB structures andMolecular modeling and deposited the resulting structure to PDB (code 1Z04).It is notable that both the modeled structureOf the LacI-DNA complex, as well as the experimental structures,Include only disjoint protein-bound DNA segments, not connected byA DNA loop.
The Head Groups Absorb All The Strain Arising From The DNA LoopThe change in the loop structure between the beginning (yellow) and the end(green) of the simulation accounts for the experimentally observed changeIn the distance between the fluorophores attached near the ends of the protein-bound DNA segments.During the simulation, the protein domains behaved very much likeRigid bodies moving with respect to each other. Thus the changes inThe protein structure occurring under the strain of the bent DNA on theSimulation time scale could be observed. The force of the loopResistance to bending was included in the MD simulations via specialForce terms acting on the protein-bound segments. TheBoundaries of the rod were defined by the instantaneous positions ofThe ends of the protein-bound operator segments. The molecular dynamics (MD) of that systemConnecting the protein-bound segment was described using a mathematical modelWhich approximated the DNA by an electrically charged elastic rod. In short, the all-atom model of the protein withThe DNA segments bound to the head groups was solvated in a bathOf water and ions.
The loop structure was much relaxedDuring the simulation, its energy dropped from ~20kT to ~12kT. In contrast, the cleft between the LacI dimers showedOnly an insignificant increase. The head groups rotated inDifferent directions with respect to their dimer core domains, as shownOn the left. In the simulations that did includeThe forces from the loop, the domain motions increased in scale,Especially those of the head groups.
X Ray Crystallography Dna Movie Of The
Three groups of interacting residues keep LacI in the "V"Configuration when wrestling the DNA loop.The first interaction (orange in figure) is the attraction betweenThe adjacent ends of the two alpha helices connecting the dimers. The figure below showsSnapshots of the protein during the cleft-opening simulation.The lock of LacI. The goal was to understand whatInteractions kept the lacI dimers in their initial "V" configuration,Preventing an opening of the cleft between them. In another simulation, we forced the opening of theProtein by applying extra forces to the ends of the protein-bound DNA.This forced pulling mimicked single molecule experiments such as atomicForce microscopy or optical tweezers. We could measure a very similarIncrease in distance in our simulation (see Figure on the right), thusExplaining the experimental observations without invoking any significantWatch a movie of the simulation here (Quicktime required,) Opening LacI: The Protein LockThe results of the multiscale simulation described above wereUnexpected: the protein "hands" did not open easily, contrary to whatHad been thought. This is a very interesting finding,Because up to now it was believed that it would be easy to open theProtein by rotating its dimer "hands" around the 4-helix bundle,Making such rotation the principal degree of freedom of the protein.Beautiful experiment measured the distance betweenFluorophores attached to the DNA loop near the lac repressorHead groups to be ~5nm larger than the same distance in the crystal structure.
Proceedings of the National Academy of Sciences, USA, 102:6783-6788, 2005. Publications Publications Database Structural dynamics of the Lac repressor-DNA complex revealed by a multiscale simulation.Elizabeth Villa, Alexander Balaeff, and Klaus Schulten. TheMutations of the key residues shall weaken the protein lock and lowerThe force barrier required to open the cleft in the micromanipulationWatch a movie of opening the protein "lock" here (Quicktime required,By Trish Barker in NCSA's Access Magazine. During theOpening of the cleft, the dimers gradually move away from each other,Causing R351 to switch to the helix in its own dimer.These interactions unravel a mystery surrounding LacI dynamics. The salt bridges are very hard to break, and contribute to theFinally, R351:1A interacts with the dipole moment of a helix fromEither the other dimer (green) or its own dimer (purple). These salt bridges do not formUnless LacI is subject to strain thereby yielding a small pre-opening ofThe cleft.
Structural basis for cooperative DNA binding by CAP and Lac repressor.Alexander Balaeff, L. Mahadevan, and Klaus Schulten. Philosophical Transactions of the Royal Society of London A. (Mathematical, Physical and Engineering Sciences), 362:1355-1371, 2004. Koudella, L. Mahadevan, and Klaus Schulten. Modeling DNA loops using continuum and statistical mechanics.Alexander Balaeff, Christophe R. Multiscale Modeling and Simulation, 2:527-553, 2004.
InvestigatorsPage created and maintained by Elizabeth Villa. Physical Review Letters, 83:4900-4903, 1999. Elastic rod model of a DNA loop in the lac operon.Alexander Balaeff, L. Mahadevan, and Klaus Schulten.
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