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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
Published on: July 6, 2012
Concentration-dependent effects of natural polyamines on peptide chain initiation and elongation in a cell-free
T Giannakouros1, H Nikolakaki, J G Georgatsos
1Laboratory of Biochemistry, School of Chemistry, University of Thessaloniki, Greece.
Abstract:
Spermidine and spermine at submillimolar concentrations stimulate the rate of incorporation of amino acid into protein in a cell-free system, directed either by endogenous or exogenous mRNA (TMV, globin). The stimulatory effects of these polyamines are exerted at both the stages of initiation and elogation and are more pronounced in the case of TMV or globin mRNA, amounting to approximately 2.3-fold stimulation over the polyamine-free system. The number of polysomes and the polysome-associated radioactivity increase approximately 2-fold in the presence of spermine. Synthesis of large polypeptides is a characteristic feature of the stimulatory event. However, elevated concentrations of spermidine and spermine strongly inhibit amino acid incorporation into protein. Inhibition is manifest at the stage of peptide elongation. In the case of endogenous mRNA the addition of an excess of polyamines results in a non uniform inhibition of amino acid incorporation. A most interesting finding is that, with increasing concentrations of polyamines, the intensity of four bands with Mr values of 63000, 44000, 15500 and 12500 respectively, increases or leastwise remains constant while others fade, indicating differential translation of proteins in the presence of polyamines.
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