Probing anomalous structural features in polypurine tract-containing RNA-DNA hybrids with neomycin B.
During (-)-strand DNA synthesis in retroviruses and Saccharomyces cerevisiae LTR retrotransposons, a purine rich region of the RNA template, known as the polypurine tract (PPT), is resistant to RNase H-mediated hydrolysis and subsequently serves as a primer for (+)-strand, DNA-dependent DNA synthesis. Although HIV-1 and Ty3 PPT sequences share no sequence similarity beyond the fact that both include runs of purine ribonucleotides, it has been suggested that these PPTs are processed by their cognate reverse transcriptases (RTs) through a common molecular mechanism. Here, we have used the aminoglycoside neomycin B (NB) to examine which structural features of the Ty3 PPT contribute to specific recognition and processing by its cognate RT. Using high-resolution NMR, direct infusion FTICR mass spectrometry, and isothermal titration calorimetry, we show that NB binds preferentially and selectively adjacent to the Ty3 3' PPT-U3 cleavage junction and in an upstream 5' region where the thumb subdomain of Ty3 RT putatively grips the substrate. Regions highlighted by NB on the Ty3 PPT are similar to those previously identified on the HIV-1 PPT sequence that are implicated as contact points for substrate binding by its RT. Our findings thus support the notion that common structural features of lentiviral and LTR-retrotransposon PPTs facilitate the interaction with their cognate RT.
Brinson
Robert G
RG
Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Turner
Kevin B
KB
Yi-Brunozzi
Hye Young
HY
Le Grice
Stuart F J
SF
Fabris
Daniele
D
Marino
John P
JP
D001483:Base Sequence; D002151:Calorimetry; D004247:DNA / Q000737:chemistry*; D005601:Framycetin / Q000737:chemistry*; D013058:Mass Spectrometry; D015335:Molecular Probes; D019906:Nuclear Magnetic Resonance, Biomolecular; D009693:Nucleic Acid Hybridization; D011687:Purines / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
D015335:Molecular Probes; D011687:Purines; D005601:Framycetin; D012313:RNA; D004247:DNA