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 reve rse transcriptase (P protein) of duck hepatitis B virus (DHBV) as an e xample, 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-labe led, partially hydrolyzed transcripts that bound to in vitro translate d His-tagged P protein were isolated using immobilized Ni2+-ions. Size analysis by PAGE is consistent with a gradual gain in binding-compete nce 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 con venient and precise fine mapping of RNA-protein interactions.