Altering the RNA-binding Mode of the U1A RBD1 Protein

Scott A. Showalter, Kathleen B. Hall

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

The N-terminal RNA-binding domain (RBD1) of the human U1A protein is evolutionarily designed to bind its RNA targets with great affinity and specificity. The physical mechanisms that modulate the coupling (local cooperativity) among amino acid residues on the extensive binding surface of RBD1 are investigated here, using mutants that replace a highly conserved glycine residue. This glycine residue, at the strand/loop junction of β3/loop3, is found in U1A RBD1, and in most RBD domains, suggesting it has a specific role in modulation of RNA binding. Here, two RBD1 proteins are constructed in which that residue (Gly53) is replaced by either alanine or valine. These new proteins are shown by NMR methods and molecular dynamics simulations to be very similar to the wild-type RBD1, both in structure and in their backbone dynamics. However, RNA-binding assays show that affinity for the U1 snRNA stem-loop II RNA target is reduced by nearly 200-fold for the RBD1-G53A protein, and by 1.6×104-fold for RBD1-G53V. The mode of RNA binding by RBD1-G53A is similar to that of RBD1-WT, displaying its characteristic non-additive free energies of base recognition and its salt-dependence. The binding mode of RBD1-G53V is altered, having lost its salt-dependence and displaying site-independence of base recognition. The molecular basis for this alteration in RNA-binding properties is proposed to result from the inability of the RNA to induce a change in the structure of the free protein to produce a high-affinity complex.

Original languageEnglish (US)
Pages (from-to)465-480
Number of pages16
JournalJournal of Molecular Biology
Volume335
Issue number2
DOIs
StatePublished - Jan 9 2004

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RNA
Glycine
Proteins
Salts
Valine
Molecular Dynamics Simulation
Alanine
U1A protein
Amino Acids

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Cite this

Showalter, Scott A. ; Hall, Kathleen B. / Altering the RNA-binding Mode of the U1A RBD1 Protein. In: Journal of Molecular Biology. 2004 ; Vol. 335, No. 2. pp. 465-480.
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Altering the RNA-binding Mode of the U1A RBD1 Protein. / Showalter, Scott A.; Hall, Kathleen B.

In: Journal of Molecular Biology, Vol. 335, No. 2, 09.01.2004, p. 465-480.

Research output: Contribution to journalArticle

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