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  Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Rothwell, P. J., Berger, S., Kensch, O., Felekyan, S., Antonik, M., Wöhrl, B. M., et al. (2003). Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proceedings of the National Academy of Sciences of the USA, 100(4): 1, pp. 1655-1660. Retrieved from http://dx.doi.org/10.1073/pnas.0434003100.

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Genre: Journal Article
Alternative Title : Proc. Natl. Acad. Sci. U. S. A.

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 Creators:
Rothwell, Paul James1, Author
Berger, Sylvia1, Author
Kensch, Oliver1, Author
Felekyan, Suren1, Author
Antonik, Matthew1, Author
Wöhrl, Birgitta Maria2, Author              
Restle, Tobias2, Author              
Goody, Roger S.2, Author              
Seidel, Claus A. M.1, Author
Affiliations:
1Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753286              
2Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753289              

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 Abstract: By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid- binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Angstrom shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.

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Language(s): eng - English
 Dates: 2003-02-18
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 12719
URI: http://dx.doi.org/10.1073/pnas.0434003100
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Title: Proceedings of the National Academy of Sciences of the USA
  Alternative Title : Proc. Natl. Acad. Sci. U. S. A.
Source Genre: Journal
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Pages: - Volume / Issue: 100 (4) Sequence Number: 1 Start / End Page: 1655 - 1660 Identifier: -