A sensitive procedure for mapping the boundaries of RNA elements binding in vitro translated proteins defines a minimal hepatitis B virus encapsidation signal.

AUTOR(ES)

Beck, J

RESUMO

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.

Documentos Relacionados

• Molecular cloning of a rat chromosome putative recombinogenic sequence homologous to the hepatitis B virus encapsidation signal.
• Mutations in the pre-core region of hepatitis B virus serve to enhance the stability of the secondary structure of the pre-genome encapsidation signal.
• A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis.
• The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function.
• Distinct requirements for primary sequence in the 5'- and 3'-part of a bulge in the hepatitis B virus RNA encapsidation signal revealed by a combined in vivo selection/in vitro amplification system.