A sensitive procedure for mapping the boundaries of RNA elements binding in vitro translated proteins defines a minimal hepatitis B virus encapsidation signal.
Using the structured RNA encapsidation signal (D(epsilon)) and the reverse transcriptase (P protein) of duck hepatitis B virus (DHBV) as an example, we devised a sensitive mapping procedure that yields accurate information on the minimal RNA sequence required for interaction with a few nanograms of an RNA-binding protein. RNAs from pools of end-labeled, partially hydrolyzed transcripts that bound to in vitro translated His-tagged P protein were isolated using immobilized Ni2+-ions. Size analysis by PAGE is consistent with a gradual gain in binding-competence from a minimum of 5 to a maximum of 8 base pairs in the basal stem of D(epsilon). The procedure should be generally applicable to the convenient and precise fine mapping of RNA-protein interactions.
Beck
J
J
Zentrum für Molekulare Biologie, University of Heidelberg, Germany.
Nassal
M
M
D015611:Hepatitis B Virus, Duck / Q000235:genetics* / Q000502:physiology; D011485:Protein Binding; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics
D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase