Dynamic binding orientations direct activity of HIV reverse transcriptase.
The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.
Abbondanzieri
Elio A
EA
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Bokinsky
Gregory
G
Rausch
Jason W
JW
Zhang
Jennifer X
JX
Le Grice
Stuart F J
SF
Zhuang
Xiaowei
X
D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D031541:Fluorescence Resonance Energy Transfer; D006678:HIV / Q000201:enzymology* / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D008024:Ligands; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D048348:Reverse Transcription; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D008024:Ligands; C034477:RNA primers; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase