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- W1978127515 abstract "Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A·U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions. Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A·U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions. ribonucleoprotein RNA recognition motif To execute their widely differing functions, RNA-binding proteins must be able to bind to their correct RNA targets with appropriate kinetics, affinities, and specificities (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar). In contrast to most DNA-binding proteins, which are presented with a double-stranded B-form helix of uniform structure in which bases can be contacted through the major groove, RNA-binding proteins must be able to bind targets with widely differing structures. Because the steep and narrow groove of double-stranded RNA does not provide proteins easy access to the bases for sequence-specific recognition, most RNA-binding proteins recognize single-stranded regions or distorted double-stranded regions in which the major groove has been widened by bulges, hairpins, or loops (2Draper D.E. J. Mol. Biol. 1999; 293: 255-270Crossref PubMed Scopus (341) Google Scholar). The natural variety of RNA targets is bound by a limited collection of RNA-binding motifs (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar, 2Draper D.E. J. Mol. Biol. 1999; 293: 255-270Crossref PubMed Scopus (341) Google Scholar). The most common of these motifs is the ribonucleoprotein (RNP)1consensus domain or the RNA-binding domain, also referred to as the RNA recognition motif (RRM). This motif is characterized by two conserved stretches of eight and six amino acid residues (RNP-1 and RNP-2) and a β-α-β-β-α-β secondary structure (see Fig.1 A) (3Nagai K. Oubridge C. Ito N. Avis J. Evans P. Trends Biochem. Sci. 1995; 20: 235-240Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 4Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar). RRMs fold into a baseball glove-like structure in which the β-sheet and the surrounding regions form the RNA binding surface. Proteins containing one or more RRMs recognize a variety of RNA sequences and structures (3Nagai K. Oubridge C. Ito N. Avis J. Evans P. Trends Biochem. Sci. 1995; 20: 235-240Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 4Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar). An RRM that binds very tightly to its RNA target is the N-terminal RRM of the spliceosomal protein U1A, which binds to an RNA hairpin in the U1 small nucleolar (sn) RNP (U1 hairpin (hp) II or U1hpII) (see Fig. 1).The U1A/U1hpII interaction has been used as a paradigm for RNA binding by a single RRM and has been the subject of a multitude of biochemical and structural analyses (4Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar). Despite these extensive studies, little is known to date about the mechanism and kinetics of this protein/RNA interaction. Using the previously solved structure of the U1A·U1hpII complex, we have engineered a series of mutants designed to individually examine the roles of electrostatics, hydrogen bonding, aromatic stacking, and RNA loop length, all of which have been implicated in formation of the U1A·U1hpII complex (5–16). The effects of these mutations on the binding dynamics were studied using a surface plasmon resonance-based biosensor (BIACORE), which permits the real-time monitoring of complex formation and dissociation (17Morton T.A. Myszka D.G. Methods Enzymol. 1998; 295: 268-294Crossref PubMed Scopus (267) Google Scholar, 18Myszka D.G. J. Mol. Recognit. 1999; 12: 1-6Crossref PubMed Scopus (35) Google Scholar, 19Myszka D.G. Methods Enzymol. 2000; 323: 325-340Crossref PubMed Scopus (188) Google Scholar). Our analyses show that complex formation occurs by two clearly distinguishable steps. First, well placed positively charged residues on the protein allow it to rapidly associate with the RNA. Next, close-range interactions at the RNA/protein interface allow the formation of a very stable complex. Together, these steps result in the high affinity of U1A for its U1 hairpin II RNA target (K D ∼32 pm). A similar two-step mechanism may play a role in many high affinity RNA/protein interactions. To execute their widely differing functions, RNA-binding proteins must be able to bind to their correct RNA targets with appropriate kinetics, affinities, and specificities (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar). In contrast to most DNA-binding proteins, which are presented with a double-stranded B-form helix of uniform structure in which bases can be contacted through the major groove, RNA-binding proteins must be able to bind targets with widely differing structures. Because the steep and narrow groove of double-stranded RNA does not provide proteins easy access to the bases for sequence-specific recognition, most RNA-binding proteins recognize single-stranded regions or distorted double-stranded regions in which the major groove has been widened by bulges, hairpins, or loops (2Draper D.E. J. Mol. Biol. 1999; 293: 255-270Crossref PubMed Scopus (341) Google Scholar). The natural variety of RNA targets is bound by a limited collection of RNA-binding motifs (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar, 2Draper D.E. J. Mol. Biol. 1999; 293: 255-270Crossref PubMed Scopus (341) Google Scholar). The most common of these motifs is the ribonucleoprotein (RNP)1consensus domain or the RNA-binding domain, also referred to as the RNA recognition motif (RRM). This motif is characterized by two conserved stretches of eight and six amino acid residues (RNP-1 and RNP-2) and a β-α-β-β-α-β secondary structure (see Fig.1 A) (3Nagai K. Oubridge C. Ito N. Avis J. Evans P. Trends Biochem. Sci. 1995; 20: 235-240Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 4Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar). RRMs fold into a baseball glove-like structure in which the β-sheet and the surrounding regions form the RNA binding surface. Proteins containing one or more RRMs recognize a variety of RNA sequences and structures (3Nagai K. Oubridge C. Ito N. Avis J. Evans P. Trends Biochem. Sci. 1995; 20: 235-240Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 4Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar). An RRM that binds very tightly to its RNA target is the N-terminal RRM of the spliceosomal protein U1A, which binds to an RNA hairpin in the U1 small nucleolar (sn) RNP (U1 hairpin (hp) II or U1hpII) (see Fig. 1). The U1A/U1hpII interaction has been used as a paradigm for RNA binding by a single RRM and has been the subject of a multitude of biochemical and structural analyses (4Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar). Despite these extensive studies, little is known to date about the mechanism and kinetics of this protein/RNA interaction. Using the previously solved structure of the U1A·U1hpII complex, we have engineered a series of mutants designed to individually examine the roles of electrostatics, hydrogen bonding, aromatic stacking, and RNA loop length, all of which have been implicated in formation of the U1A·U1hpII complex (5–16). The effects of these mutations on the binding dynamics were studied using a surface plasmon resonance-based biosensor (BIACORE), which permits the real-time monitoring of complex formation and dissociation (17Morton T.A. Myszka D.G. Methods Enzymol. 1998; 295: 268-294Crossref PubMed Scopus (267) Google Scholar, 18Myszka D.G. J. Mol. Recognit. 1999; 12: 1-6Crossref PubMed Scopus (35) Google Scholar, 19Myszka D.G. Methods Enzymol. 2000; 323: 325-340Crossref PubMed Scopus (188) Google Scholar). Our analyses show that complex formation occurs by two clearly distinguishable steps. First, well placed positively charged residues on the protein allow it to rapidly associate with the RNA. Next, close-range interactions at the RNA/protein interface allow the formation of a very stable complex. Together, these steps result in the high affinity of U1A for its U1 hairpin II RNA target (K D ∼32 pm). A similar two-step mechanism may play a role in many high affinity RNA/protein interactions. We thank Ian Haworth, Huynh-Hoa Bui, Meline Bayramyan, and Peter Laird for useful comments and help with the structure analysis and figures, and we thank members of the Laird-Offringa laboratory for helpful criticism." @default.
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- W1978127515 title "Two Functionally Distinct Steps Mediate High Affinity Binding of U1A Protein to U1 Hairpin II RNA" @default.
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