Specific RNA binding by a single C2H2 zinc finger.
Zinc finger proteins with high affinity for human immunodeficiency virus Rev responsive element stem loop IIB (RRE-IIB) were previously isolated from a phage display zinc finger library. Zinc fingers from one of these proteins, RR1, were expressed individually and assayed for RRE-IIB affinity. The C-terminal zinc finger retained much of the binding affinity of the two-finger parent and was disrupted by mutations predicted to narrow the RRE-IIB major groove and which disrupt Rev binding. In contrast, the N-terminal zinc finger has a calculated affinity at least 1000-fold lower. Despite the high affinity and specificity of RR1 for RRE-IIB, binding affinity for a 234-nucleotide human immunodeficiency virus Rev responsive element (RRE234) was significantly lower. Therefore, zinc finger proteins that bind specifically to RRE234 were constructed using an in vitro selection and recombination approach. These zinc fingers bound RRE234 with subnanomolar dissociation constants and bound the isolated RRE-IIB stem loop with an affinity 2 orders of magnitude lower but similar to the affinity of an arginine-rich peptide derived from Rev. These data show that single C2H2 zinc fingers can bind RNA specifically and suggest that their binding to stem loop IIB is similar to that of Rev peptide. However, binding to RRE234 is either different from stem loop IIB binding or the tertiary structure of stem loop IIB is changed within the Rev responsive element.
Friesen
W J
WJ
Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Darby
M K
MK
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers
D016601:RNA-Binding Proteins; D012313:RNA