A Sensitive Procedure for Mapping the Boundaries of RNA Elements Binding in vitro Translated Proteins Defines a Minimal Hepatitis B Virus Encapsidation Signal

摘要

Using the structured RNA encapsidation signal (De) 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 Dε. The procedure should be generally applicable to the convenient and precise fine mapping of RNA–protein interactions.