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

@article{citeulike:2632700, 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-A 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.}, address = {Abteilung Spektroskopie und Photochemische Kinetik, Max-Planck-Institut f\"{u}r Biophysikalische Chemie, Am Fassberg 11, D-37077 G\"{o}ttingen, Germany.}, author = {Rothwell, P. J. and Berger, S. and Kensch, O. and Felekyan, S. and Antonik, M. and W\"{o}hrl, B. M. and Restle, T. and Goody, R. S. and Seidel, C. A. }, citeulike-article-id = {2632700}, doi = {10.1073/pnas.0434003100}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {fret, origami}, month = {February}, number = {4}, pages = {1655--1660}, posted-at = {2008-04-21 16:00:52}, priority = {2}, title = {Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes.}, url = {http://dx.doi.org/10.1073/pnas.0434003100}, volume = {100}, year = {2003} }

TY - JOUR ID - citeulike:2632700 L3 - citeulike-article-id:2632700 TI - Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. JF - Proceedings of the National Academy of Sciences of the United States of America VL - 100 IS - 4 SP - 1655 EP - 1660 AD - Abteilung Spektroskopie und Photochemische Kinetik, Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany. SN - 0027-8424 N2 - 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-A 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. KW - fret KW - origami AU - Rothwell, PJ AU - Berger, S AU - Kensch, O AU - Felekyan, S AU - Antonik, M AU - Wöhrl, BM AU - Restle, T AU - Goody, RS AU - Seidel, CA PY - 2003/02/18/ UR - http://dx.doi.org/10.1073/pnas.0434003100 ER -