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.
journal_name
Proc Natl Acad Sci U S Aauthors
Rothwell PJ,Berger S,Kensch O,Felekyan S,Antonik M,Wöhrl BM,Restle T,Goody RS,Seidel CAdoi
10.1073/pnas.0434003100keywords:
subject
Has Abstractpub_date
2003-02-18 00:00:00pages
1655-60issue
4eissn
0027-8424issn
1091-6490pii
0434003100journal_volume
100pub_type
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