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.
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
I A
IA
Laird-Offringa
J G
JG
Belasco
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