Polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus IRES structure via binding multiple sites in a unique orientation.
Polypyrimidine tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3' end, and RBD3 contacting the 5' end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES.
Kafasla
Panagiota
P
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
Morgner
Nina
N
Pöyry
Tuija A A
TA
Curry
Stephen
S
Robinson
Carol V
CV
Jackson
Richard J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D004686:Encephalomyocarditis virus / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment
D012367:RNA, Viral; D038941:Polypyrimidine Tract-Binding Protein