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Nucleic Acids Res. 2002 Nov 15;30(22):4855-63.

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

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

Abstract

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates DNA synthesis from the 3' end of human tRNA(Lys3). We have used cis-acting hammerhead ribozymes to produce homogeneous-length transcribed tRNA(Lys3) 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 tRNA(Lys3), 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 tRNA(Lys3) to grow.

PMID:
12433988
PMCID:
PMC137168
DOI:
10.1093/nar/gkf620
[Indexed for MEDLINE]
Free PMC Article

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