Analysis of RNA-binding proteins by in vitro genetic selection: identification of an amino acid residue important for locking U1A onto its RNA target.
An in vitro genetic system was developed as a rapid means for studying the specificity determinants of RNA-binding proteins. This system was used to investigate the origin of the RNA-binding specificity of the mammalian spliceosomal protein U1A. The U1A domain responsible for binding to U1 small nuclear RNA was locally mutagenized and displayed as a combinatorial library on filamentous bacteriophage. Affinity selection identified four U1A residues in the mutagenized region that are important for specific binding to U1 hairpin II. One of these residues (Leu-49) disproportionately affects the rates of binding and release and appears to play a critical role in locking the protein onto the RNA. Interestingly, a protein variant that binds more tightly than U1A emerged during the selection, showing that the affinity of U1A for U1 RNA has not been optimized during evolution.
Laird-Offringa
I A
IA
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Belasco
J G
JG
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D015723:Gene Library; D014644:Genetic Variation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic; D013329:Structure-Activity Relationship
D004274:DNA, Recombinant; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein