Chemical shift mapping of RNA interactions with the polypyrimidine tract binding protein.
The polypyrimidine tract binding protein (PTB), a homodimer that contains four RRM-type RNA binding domains per monomer, plays important roles in both the regulation of alternative splicing and the stimulation of translation initiation as directed by the internal ribosome entry sites of certain picornaviruses. We have used chemical shift mapping experiments to probe the interactions between PTB-34, a recombinant fragment that contains the third and fourth RRM domains of the protein, and a number of short pyrimidine-rich RNA oligonucleotides. The results confirm that the RNAs interact primarily with the beta-sheet surface of PTB-34, but also reveal roles for the two long flexible linkers within the protein fragment, a result that is supported by mutagenesis experiments. The mapping indicates distinct binding preferences for RRM3 and RRM4 with the former making a particularly specific interaction with the sequence UCUUC.
Yuan
Xuemei
X
Wolfson Laboratory, Exhibition Road, London SW7 2AY, UK.
Davydova
Natalia
N
Conte
Maria R
MR
Curry
Stephen
S
Matthews
Stephen
S
D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D001059:Apoproteins; D009843:Oligoribonucleotides; D010446:Peptide Fragments; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA