関: shadowing

WordNet

the act of following someone secretly (同)tailing

English Journal

Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8.

Makhov AM, Boehmer PE, Lehman IR, Griffith JD.Author information Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599-7295, USA.AbstractUL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers. When the binding of UL9 protein to double strand (ds) DNA containing ss tails was examined by shadowcasting methods, UL9 protein was seen bound to the ss tails or ss/ds junctions; addition of ATP led to its appearance internally along the ds segment. When UL9 protein and ICP8 were incubated together with the tailed dsDNA in the presence of ATP, a highly ordered unwinding of the DNA was observed by negative staining that appeared to progress through four distinct stages: (1) binding of ICP8 to the ss tail and progressive coverage of the ds portion by UL9 protein; (2) formation of highly condensed regular filaments; (3) relaxation of the condensed structures into coiled-coils; and (4) unwinding of the coils and release of ICP8-covered linear ssDNAs. This process represents a mechanism of unwinding that is very different from ones that proceed by a progressive unwinding at Y-shaped forks that move along the DNA.
Journal of molecular biology.J Mol Biol.1996 May 24;258(5):789-99.
UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding prote
PMID 8637010

Visualization of TBP oligomers binding and bending the HIV-1 and adeno promoters.

Griffith JD, Makhov A, Zawel L, Reinberg D.Author information Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295.AbstractThe binding of the 28 kDa yeast TATA binding protein (yTBP) to the HIV and adeno major late promoters has been examined by electron microscopy (EM). Three different EM preparative methods were employed: direct mounting and shadowcasting of fixed samples, cryofixation and freeze-drying followed by shadowcasting, and negative staining of unfixed samples. Excellent agreement among the three methods was obtained. With ten yTBP monomers/DNA fragment, up to 25% of the DNA molecules contained easily distinguished protein particles at the TATA box and, less frequently, smaller particles were observed. Non-specific binding to DNA ends was common. The mass of the easily distinguished particles measured 63(+/- 5) kDa (cryofixation and shadowcasting) and 48(+/- 6) kDa (negative staining) indicating TBP dimerization. With 22 and 44 yTBP monomers/DNA, yTBP polymerization produced DNA-protein rods 9 nm wide and 20 to 30 nm long, frequently with two DNA strands exiting one end. Bending analysis revealed that yTBP dimers bend the DNA about the TATA box by 80 to 90 degrees. Although these protein ratios are relatively high, the structures formed demonstrate the propensity of yTBP to engage in protein-protein interactions.
Journal of molecular biology.J Mol Biol.1995 Mar 10;246(5):576-84.
The binding of the 28 kDa yeast TATA binding protein (yTBP) to the HIV and adeno major late promoters has been examined by electron microscopy (EM). Three different EM preparative methods were employed: direct mounting and shadowcasting of fixed samples, cryofixation and freeze-drying followed by sh
PMID 7533216

Visualization of a tertiary structural domain of the Tetrahymena group I intron by electron microscopy.

Wang YH, Murphy FL, Cech TR, Griffith JD.Author information Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295.AbstractThe P4-P6 domain RNA of the group I intron of Tetrahymena thermophila has previously been shown by chemical probing to be an independently folding domain of the intron's tertiary structure. To directly visualize this tertiary structure, the P4-P6 domain and two folding defective mutants were prepared for high-resolution electron microscopy using tungsten shadowcasting. In the presence of Mg2+, the P4-P6 domain predominantly consists of compact molecules, while the two mutant RNAs are nearly all rod-like molecules. The measured length of the rod-like molecules is 64 (+/- 6) bp, which agrees closely with the length expected for molecules containing secondary structure only. In the absence of Mg2+, the P4-P6 domain contains threefold or tenfold fewer compact structures (depending on the mounting procedures) than in the presence of Mg2+. These results provide direct evidence for the overall shape of the tertiary structure proposed on the basis of biochemical experiment, and they confirm the Mg2+ dependence of tertiary folding. An equilibrium between the extended (rod-like) and the compact structures is suggested, with the concentration of bound Mg2+ and different mounting methods influencing the direction of the equilibrium. The entire group I ribozyme (L-21 Sca I RNA) was also examined by electron microscopy in the presence of Mg2+, and was revealed to have a compact shape. These studies present a direct demonstration of long-range interactions in a catalytic RNA molecule.
Journal of molecular biology.J Mol Biol.1994 Feb 11;236(1):64-71.
The P4-P6 domain RNA of the group I intron of Tetrahymena thermophila has previously been shown by chemical probing to be an independently folding domain of the intron's tertiary structure. To directly visualize this tertiary structure, the P4-P6 domain and two folding defective mutants were prepare
PMID 7508985