A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.
A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.
San Diego Institute of Molecular Biology and Structural RNA, CA 92121.
H N
HN
Rubin
M N
MN
Halim
P C
PC
Leavis
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D005453:Fluorescence; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000378:metabolism; D005914:Globins / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D039102:Poly(A)-Binding Proteins; D011119:Polynucleotides / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D005656:Fungal Proteins; D010455:Peptides; D039102:Poly(A)-Binding Proteins; D011119:Polynucleotides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D005914:Globins