Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (227K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Pata, J. D.
Right arrow Articles by Steitz, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pata, J. D.
Right arrow Articles by Steitz, T. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2002, Vol. 30, No. 22 4855-4863
© 2002 Oxford University Press

Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex

Janice D. Pata1, Bradford R. King1,3 and Thomas A. Steitz*,1,2,3

1 Department of Molecular Biophysics and Biochemistry, 2 Department of Chemistry and 3 Howard Hughes Medical Institute, Yale University, New Haven, CT 06520-8114, USA

*To whom correspondence should be addressed at Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA. Tel: +1 203 432 5619; Fax: +1 203 432 3282; Email: eatherton{at}csb.yale.edu
Present address:
Bradford R. King, Rib-X Pharmaceuticals, New Haven, CT 06511, USA

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates DNA synthesis from the 3' end of human tRNALys3. We have used cis-acting hammerhead ribozymes to produce homogeneous-length transcribed tRNALys3 and have developed conditions for purifying highly structured RNAs on a modified tube-gel apparatus. Titration experiments show that this RNA can assemble into an initiation complex that contains equimolar amounts of HIV-1 RT, transcribed tRNALys3, and chemically synthesized template RNA. We have purified this complex using gel-filtration chromatography and have found that it is homogeneous with respect to molecular weight, demonstrating that the initiation complex forms a single discrete species at micromolar concentrations. When this initiation complex is supplied with deoxynucleotides, essentially all of the tRNA is used as a primer by HIV-1 RT and is fully extended to the 5' end of the template. Thus, in vitro transcribed tRNA can be used efficiently as a primer by HIV-1 RT. We have also obtained crystals of the HIV-1 initiation complex that require the precisely defined ends of this in vitro transcribed tRNALys3 to grow.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
J. T. Miller, A. Khvorova, S. A. Scaringe, and S. F. J. Le Grice
Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes
Nucleic Acids Res., September 1, 2004; 32(15): 4687 - 4695.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. D. Pata, W. G. Stirtan, S. W. Goldstein, and T. A. Steitz
Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors
PNAS, July 20, 2004; 101(29): 10548 - 10553.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.