Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase.
A highly efficient cell-free translation system has been combined with suppressor tRNA technology to substitute nor-Tyr and 3-fluoro-Tyr in place of Tyr183 at the DNA polymerase active site of p66 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Supplementing the wild-type HIV-1 p51 RT subunit into this translation system permitted reconstitution of the biologically relevant p66/p51 heterodimer harboring Tyr analogs exclusively on the catalytically competent p66 subunit. Addition of an affinity tag at the p66 C-terminus allowed rapid, one-step purification of reconstituted and selectively mutated heterodimer HIV-1 RT via strep-Tactin-agarose affinity chromatography. The purified enzyme was demonstrated to be free of contaminating nucleases, allowing characterization of the DNA polymerase and ribonuclease H activities associated with HIV-1 RT. Preliminary characterization of HIV-1 RT(nor-Tyr) and HIV-1 RT(m-fluoro-Tyr) is presented. The success of this strategy will facilitate detailed molecular analysis of structurally and catalytically critical amino acids via their replacement with closely related, unnatural analogs.
Klarmann
George J
GJ
HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, MD, USA.
Eisenhauer
Brian M
BM
Zhang
Yi
Y
Sitaraman
Kalavathy
K
Chatterjee
Deb K
DK
Hecht
Sidney M
SM
Le Grice
Stuart F J
SF
D019943:Amino Acid Substitution; D002474:Cell-Free System; D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008954:Models, Biological; D021122:Protein Subunits / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry / Q000302:isolation & purification; D016914:Ribonuclease H / Q000737:chemistry / Q000302:isolation & purification
D021122:Protein Subunits; D012343:RNA, Transfer; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H