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Showing papers by "Carlos Bustamante published in 1999"


Journal ArticleDOI
TL;DR: A folding algorithm to predict the structure of an RNA from its sequence is suggested, but to solve the RNA folding problem one needs thermodynamic data on tertiary structure interactions, and identification and characterization of metal-ion binding sites.

910 citations


Journal ArticleDOI
TL;DR: Observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location, and by using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

245 citations


Journal ArticleDOI
TL;DR: The polymerization of individual RecA-DNA filaments was followed in real time, and their mechanical properties were characterized with force-measuring laser tweezers, finding that the stretch modulus of a filament is dominated by its central DNA component, while its bending rigidity is controlled by its (eccentric) protein component.
Abstract: The polymerization of individual RecA–DNA filaments, containing either single-stranded or double-stranded DNA, was followed in real time, and their mechanical properties were characterized with force-measuring laser tweezers. It was found that the stretch modulus of a filament is dominated by its (central) DNA component, while its bending rigidity is controlled by its (eccentric) protein component. The longitudinal stiffness of DNA increases 6- to 12-fold when the DNA is contained in the protein helix. Both the stretch modulus and the bending rigidity of a fiber change in the presence of various nucleotide cofactors—e.g., [γ-thio]ATP, ATP, and ADP—indicating a substantial re-arrangement of spatial relationships between the nucleic acid and the protein scaffold. In particular, when complexed with ATP, a fiber becomes twice as extensible as a [γ-thio]ATP fiber, suggesting that 32% of the DNA-binding sites have been released in its core. Such release may enable easy rotation of the DNA within the protein helix or slippage of the DNA through the center of the protein helix.

227 citations


Journal ArticleDOI
TL;DR: The structural analysis of the open promoter complexes revealed that two‐thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one‐third ispart of the downstream region.
Abstract: High-resolution atomic force microscopy (AFM) and biochemical methods were used to analyze the structure of Escherichia coli RNA polymerase.sigma(70) (RNAP) open promoter complex (RP(o)). A detailed analysis of a large number of molecules shows that the DNA contour length of RP(o) is reduced by approximately 30 nm (approximately 90 bp) relative to the free DNA. The DNA bend angle measured with different methods varied from 55 to 88 degrees. The contour length reduction and the DNA bend angle were much less in inactive RNAP-DNA complexes. These results, together with previously published observations, strongly support the notion that during transcription initiation, the promoter DNA wraps nearly 300 degrees around the polymerase. This amount of DNA bending requires an energy of 60 kJ/mol. The structural analysis of the open promoter complexes revealed that two-thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one-third is part of the downstream region. Based on these data, a model of the sigma(70).RP(o) conformation is proposed.

217 citations


Journal ArticleDOI
TL;DR: The size of unligated lambda-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape.
Abstract: Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated λ-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed.

58 citations