Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65.
The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.
Sickmier
E Allen
EA
Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205, USA.
Frato
Katherine E
KE
Shen
Haihong
H
Paranawithana
Shanthi R
SR
Green
Michael R
MR
Kielkopf
Clara L
CL
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry / Q000235:genetics*; D012322:RNA Precursors / Q000096:biosynthesis*; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000378:metabolism*; D016415:Sequence Alignment; D000072668:Splicing Factor U2AF; D014529:Uridine / Q000235:genetics / Q000378:metabolism
D009687:Nuclear Proteins; D012322:RNA Precursors; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; C000607495:Zrsr1 protein, mouse; D038941:Polypyrimidine Tract-Binding Protein; D004247:DNA; D014529:Uridine