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J. Biol. Chem., Vol. 276, Issue 20, 17484-17496, May 18, 2001
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From the Howard Hughes Medical Institute and the Departments of
Genetics and Medicine, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
The KH domain mediates RNA binding in a wide
range of proteins. Here we investigate the RNA-binding properties of
two abundant RNA-binding proteins,
Optimized RNA Targets of Two Closely Related Triple KH Domain
Proteins, Heterogeneous Nuclear Ribonucleoprotein K and
CP-2KL,
Suggest Distinct Modes of RNA Recognition*
,
CP-2KL and heterogeneous nuclear
ribonucleoprotein (hnRNP) K. These proteins constitute the major
poly(C) binding activity in mammalian cells, are closely related on the
basis of the structures and positioning of their respective triplicated KH domains, and have been implicated in a variety of
post-transcriptional controls. By using SELEX, we have obtained sets of
high affinity RNA targets for both proteins. The primary and secondary
structures necessary for optimal protein binding were inferred in each
case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for CP-2KL and hnRNP K were
both enriched for cytosine bases and were presented in a
single-stranded conformation. In contrast to these shared
characteristics, the optimal target sequence for hnRNP K is composed of
a single short "C-patch" compatible with recognition by a single KH
domain whereas that for CP-2KL encompassed three such C-patches
suggesting more extensive interactions. The binding specificities of
the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized CP-2KL-binding site
was supported by comparison of SELEX target structure with that of the
native human -globin 3'-untranslated region. These data indicate
that modes of macromolecular interactions of arrayed KH domains can
differ even among closely related KH proteins and that binding
affinities are substantially dependent on the presentation of the
target site within the RNA secondary structure.
*
This work was supported in part by National
Institutes of Health Grants HL38632 and CA72765 (to S. A. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Novo Nordisk, Enzyme Screening, 6E2.09,
Smørmosevej 10-12, DK-2880 Bagsværd, Denmark.
§
Present address: Gen-Probe, Inc., 10210 Genetic Center Dr., San
Diego, CA.
¶
Investigator at the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Rm. 428, Clinical Research Bldg.,
415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-7834; Fax:
215-573-5157; E-mail: liebhabe@mail.med.upenn.edu.
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