RESUMO
dsRBP-ZFa is a Xenopus zinc finger protein that binds dsRNA and RNA-DNA hybrids with high affinity and in a sequence-independent manner. The protein consists of a basic N-terminal region with seven C2H2 zinc finger motifs and an acidic C-terminal region that is not required for binding. The last four zinc finger motifs, and the linkers that join them, are nearly identical repeats, while the first three motifs and their linkers are each unique. To identify which regions of the protein are involved in nucleic acid binding, we examined the ability of five protein fragments to bind dsRNA and RNA-DNA hybrids. Our studies reveal that a fragment encompassing the three N-terminal, unique zinc finger motifs and another encompassing the last three of the nearly identical motifs have binding properties similar to the full-length protein. Since these two fragments do not share zinc finger motifs of the same sequence, dsRBP-ZFa must contain more than one type of zinc finger motif capable of binding dsRNA. As with the full-length protein, ssRNA and DNA do not significantly compete for dsRNA binding by the fragments.
Assuntos
Proteínas de Ligação a DNA , Fragmentos de Peptídeos/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus , Dedos de Zinco , Sequência de Aminoácidos , Animais , Ligação Competitiva/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/síntese química , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/biossíntese , Especificidade por Substrato/genética , Xenopus , Dedos de Zinco/genéticaRESUMO
Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix.
Assuntos
Proteínas de Ligação a DNA , DNA/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas de Xenopus , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Biblioteca Gênica , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , XenopusRESUMO
Analysis of cDNA clones encoding transforming growth factor (TGF)-beta 2 predicts two different precursor proteins derived by alternative mRNA splicing; a 414 amino acid precursor [TGF-beta 2(414)] and a 442 amino acid precursor [TGF-beta 2(442)]. The two proteins differ by a 28 amino acid insertion within the pro-region of TGF-beta 2(442). In order to characterize the TGF-beta 2-related proteins encoded by the TGF-beta 2(442) cDNA and determine whether it could, in fact, direct the synthesis of active growth factor, we have expressed this gene in Chinese hamster ovary (CHO) cells and, after amplification with methotrexate, obtained stable clones secreting TGF-beta 2(442). The TGF-beta 2 secreted by these cells was latent as acidification was necessary to detect optimal biological activity. In addition to mature TGF-beta 2, high molecular weight pro-region containing proteins were also secreted as analyzed by immunoblotting using site-specific anti-peptide antibodies. These proteins migrated differently than those secreted by CHO cells transfected with cDNA encoding TGF-beta 2(414), indicating that structural differences exist between the two complexes. An anti-peptide antiserum was produced in rabbits against the 28 amino acid insert region of TGF-beta 2(442). This sera was then used to detect the presence of TGF-beta 2(442) in serum-free media conditioned by BSC-40 cells. Since the TGF-beta 2(442) precursor is produced and secreted by a non-recombinant cell line, this suggests that it may play a physiological role in regulating the activity of TGF-beta 2.
