Nuclear Magnetic resonance spectroscopy of escherichia coli tRNA (LYS) and the t RNA)LYS)-HIV RNA complex

Human tRNA(Lys,3) is the sole primer of HIV reverse transcription. The interaction between the tRNA and HIV RNA was studied by using E. coli tRNA(Lys) as a model. A reversed phase C4 method was developed for fractionation of tRNAs. This method was used to isolate (15)N labeled tRNA(Lys) from E. coli grown in minimal medium. The modification status of the isolated tRNA was verified by RNase Tl digestion followed by LC/MS. The isolated tRNA(Lys) was characterized by NMR. Data from (1)H and (1)H-(15)N spectroscopy were used to assign residues in the tRNA. An RNA construct based on the HIV Mai sequence was designed with sequence modifications to favor binding to E.coli tRNA(Lys). The construct was designated M3EC. The construct contained highly conserved elements of the viral RNA that are known to interact with the tRNA primer during reverse transcription. M3EC was transcribed in vitro from a DNA template by using rNTPs and T7 RNA polymerase. The tRNA-M3EC complex was formed by heat annealing. Specific binding of MSEC to E. coli tRNA(Lys) and complex formation was analyzed by an electrophoretic mobility shift assay. The tRNA(Lys)-M3EC complex was characterized by NMR. Residues in the complex were assigned by using data from (1)H and (1)H-(15)N HSQC experiments. Evidence for extensive interaction between the two RNA molecules was seen. Some tertiary interactions in the free tRNA were disrupted. Extensive disruption of base pairs in the acceptor and T?C stems was also observed. These bases formed new base pairs with the M3bC residues, as evidenced by appearance of new peaks m the HSQC spectrum. There was also proof of interaction between the A-loop and the tRNA anticodon. The NH11 proton of t(6)^A37 in the anticodon gave two signals in the complex, resulting from bound and free states. The change in chemical shift of the t(6)A proton indicates that modified nucleotides in the anticodon play a significant role in the A-loop/anticodon interaction. It also supports the kissing hairpin model proposed for the interaction.