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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.


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