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  Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding

Thrall, S. H., Reinstein, J., Wöhrl, B. M., & Goody, R. S. (1996). Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding. Biochemistry, 35(14), 4609-4618. doi:10.1021/bi9526387.

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Genre: Journal Article
Alternative Title : Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding

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Thrall, Sara H., Author
Reinstein, Jochen1, Author              
Wöhrl, Birgitta M., Author
Goody, Roger S.2, Author              
Affiliations:
1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              
2Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              

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Free keywords: affinity; Anticodon; dissociation; dna-synthesis; Escherichia coli; Fluorescence spectroscopy; ; HIV-1; Human-immunodeficiency-virus; Ionic-strength; Reverse Transcriptase; Tryptophan
 Abstract: A host cell-derived tRNA3Lys molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome. We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA3Lys, and to address RT's putative role in the required and specific packaging of tRNA3Lys into the budding virus. Titration of RT with tRNA3Lys resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (Kd) of 57.6 +/- 7.5 nM was derived. Titration of RT with Escherichia coli tRNA2Glu, E. coli tRNA2Tyr, E. coli tRNALys, yeast tRNAPhe, or in vitro-synthesized human tRNA3Lys (no base modifications) resulted in similar fluorescence changes and Kd values as obtained for the natural tRNA3Lys. The specific interaction between RT and tRNA3Lys during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT. Other factors during viral assembly must therefore cooperate in the packaging of tRNA3Lys. The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding. The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNAPhe was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA. The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites. Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands. The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both tRNA and p/t binding. Reverse transcriptase was found to bind to the mutant tRNA 10-fold more tightly than to the unaltered tRNA (Kd = 4.5 +/- 1.0 and 44.6 +/- 6.6 nM, respectively). Further analyses revealed that the tighter affinity is probably due to a preferred p/t binding mode and not to one expected if separate tRNA and p/t binding regions are accessed simultaneously by the same molecule.

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Language(s): eng - English
 Dates: 1996-02-051995-11-061996-04-09
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 665725
DOI: 10.1021/bi9526387
URI: https://www.ncbi.nlm.nih.gov/pubmed/8605212
Other: 6221
 Degree: -

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Title: Biochemistry
Source Genre: Journal
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Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 35 (14) Sequence Number: - Start / End Page: 4609 - 4618 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103