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A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and
H N Rubin1, M N Halim, P C Leavis
1San Diego Institute of Molecular Biology and Structural RNA, CA 92121.
Abstract:
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.
Insights
A synthesized peptide binds strongly to poly(A) tails on mRNA, enhancing protein synthesis. This peptide selectively binds adenosine triphosphate (ATP) and mimics native poly(A) binding protein function.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Poly(A) binding proteins (PABPs) are crucial for mRNA stability and translation.
- A conserved consensus sequence (GKSKGFGFV) is found in PABPs.
Purpose of the Study:
- To synthesize and characterize a peptide containing the PABP consensus sequence.
- To investigate the peptide's binding affinity to nucleic acids and its effect on mRNA translation.
Main Methods:
- Peptide synthesis and biochemical assays.
- Circular dichroism spectroscopy.
- In vitro translation assays.
Main Results:
- The 15-amino acid peptide specifically binds poly(A) and ATP.
- Circular dichroism confirmed the peptide's secondary structure is consistent with native PABPs.
- The peptide significantly enhanced in vitro translation of polyadenylated mRNA but not deadenylated mRNA.
Conclusions:
- The synthesized peptide effectively mimics PABP function by binding poly(A) tails.
- This peptide can enhance mRNA translation, highlighting the importance of the poly(A) tail in this process.