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

Nucleic Acids Res. 2002 Nov 15;30(22):4855-63. doi: 10.1093/nar/gkf620.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Chromatography, Gel
  • Crystallization
  • DNA / biosynthesis
  • HIV Reverse Transcriptase* / chemistry
  • HIV Reverse Transcriptase* / isolation & purification
  • HIV Reverse Transcriptase* / metabolism
  • Humans
  • Macromolecular Substances
  • Molecular Sequence Data
  • RNA / chemistry
  • RNA / isolation & purification
  • RNA / metabolism
  • RNA, Catalytic / metabolism
  • RNA, Transfer, Lys* / chemistry
  • RNA, Transfer, Lys* / isolation & purification
  • RNA, Transfer, Lys* / metabolism
  • Templates, Genetic
  • Transcription, Genetic


  • Macromolecular Substances
  • RNA, Catalytic
  • RNA, Transfer, Lys
  • hammerhead ribozyme
  • RNA
  • DNA
  • HIV Reverse Transcriptase