Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding.
Tethered hydroxyl-radical probing has been used to determine the orientation of binding of polypyrimidine tract-binding protein (PTB) to the poliovirus type 1 (Mahoney) (PV-1(M)) internal ribosome entry site/segment (IRES)-the question of which RNA-binding domain (RBD) binds to which sites on the IRES. The results show that under conditions in which PTB strongly stimulates IRES activity, a single PTB is binding to the IRES, a finding which was confirmed by mass spectrometry of PTB/IRES complexes. RBDs1 and 2 interact with the basal part of the Domain V irregular stem loop, very close to the binding site of eIF4G, and RBDs3 and 4 interact with the single-stranded regions flanking Domain V. The binding of PTB is subtly altered in the presence of the central domain (p50) of eIF4G, and p50 binding is likewise modified if PTB is present. This suggests that PTB stimulates PV-1(M) IRES activity by inducing eIF4G to bind in the optimal position and orientation to promote internal ribosome entry, which, in PV-1(M), is at an AUG triplet 30 nt downstream of the base of Domain V.
Kafasla
Panagiota
P
Department of Biochemistry, University of Cambridge, Cambridge, UK.
Morgner
Nina
N
Robinson
Carol V
CV
Jackson
Richard J
RJ
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D018387:Codon, Initiator / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018387:Codon, Initiator; D039603:Eukaryotic Initiation Factor-4G; D012367:RNA, Viral; D038941:Polypyrimidine Tract-Binding Protein