The interactions of the human double-stranded
RNA-binding zinc
finger protein JAZ with RNA or DNA were investigated using electrophoretic
mobility-shift assays, isothermal calorimetry, and nuclear magnetic
resonance spectroscopy. Consistent with previous reports, JAZ has
very low affinity for duplex DNA or single-stranded RNA, but it binds
preferentially to double-stranded RNA (dsRNA) with no detectable sequence
specificity. The affinity of JAZ for dsRNA is unaffected by local
structural features such as loops, overhangs, and bulges, provided
a sufficient length of reasonably well-structured A-form RNA (about
18 bp for a single zinc finger) is present. Full-length JAZ contains
four Cys2His2 zinc fingers (ZF1–4) and
has the highest apparent affinity for dsRNA; two-finger constructs
ZF12 and ZF23 have lower affinity, and ZF34 binds even more weakly.
The fourth zinc finger, ZF4, has no measurable RNA-binding affinity.
Single zinc finger constructs ZF1, ZF2, and ZF3 show evidence for
multiple-site binding on the minimal RNA. Fitting of quantitative
NMR titration and isothermal calorimetry data to a two-site binding
model gave Kd1 ∼ 10 μM and Kd2 ∼ 100 μM. Models of JAZ–RNA
complexes were generated using the high-ambiguity-driven biomolecular
docking (HADDOCK) program. Single zinc fingers bind to the RNA backbone
without sequence specificity, forming complexes with contacts between
the RNA minor groove and residues in the N-terminal β strands
and between the major groove and residues in the helix–kink–helix
motif. We propose that the non-sequence-specific interaction between
the zinc fingers of JAZ with dsRNA is dependent only on the overall
shape of the A-form RNA.
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