Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex.
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.
Pata
Janice D
JD
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
King
Bradford R
BR
Steitz
Thomas A
TA
D001483:Base Sequence; D002850:Chromatography, Gel; D003460:Crystallization; D004247:DNA / Q000096:biosynthesis; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D016337:RNA, Catalytic; D012357:RNA, Transfer, Lys; C427204:hammerhead ribozyme; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase