Biological activity of avian myeloblastosis virus RNA in cell-free systems for protein synthesis Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/2f75rc51x

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  • Avian myeloblastosis virus RNA was fractionated into a high molecular weight RNA and a low molecular weight RNA fraction by sucrose density centrifugation. The larger RNA component had a sedimentation constant of 65S and the low molecular weight RNA about 4S. The low molecular RNA has two biological properties of interest. First, it behaves like tRNA in that it will attach amino acids in the presence of aminoacyl synthetases and second, it is a potent inhibitor of in vitro protein synthesis. The properties of this RNA fraction with regard to in vitro peptide synthesis were investigated in the E. coli and chick polysome cell-free systems. Peptide synthesis using a S30 preparation from E. coli, is inhibited about 50% with 0.5 μg of virus RNA when either Qß phage RNA or E. coli RNA is used as a template. Peptide synthesis directed by the homopolymers poly U and poly A are not inhibited by the low molecular weight virus RNA. When poly C is used as a template, proline polymerization is inhibited. Excess ribosomes added to the in vitro system did not reverse the inhibition by virus RNA. Increasing either mRNA or the ribosome-free supernatant reversed the inhibition. Peptide synthesis, using chick polysome cell-free systems was inhibited to a lesser extent. The results of these studies are consistent with a mechanism by which inhibition occurs prior to the formation of the messenger-ribosome complex. The transfer activity of the low molecular weight virus RNA fraction was examined. Virus RNA charged with amino acid transferred amino acids to TCA-precipitable protein to a degree similar to that transferred by liver aminoacyl-tRNA. Low molecular weight virus RNA appears to contain an RNA fraction with properties identical to tRNA. The development of systems for protein synthesis from chick cells was described. A ribosome preparation, capable of using poly U and Qß RNA as templates for protein synthesis, contained low endogenous mRNA activity and incorporated phenylalanine linearly for 30 minutes in the presence of poly U. Results using a chick polysome cell-free system were described. The endogenous message-containing system required ATP and GTP for incorporation, was sensitive to the addition of puromycin and incorporated amino acids when added in the form of aminoacyl-tRNA. Biological activity of high molecular weight virus RNA was examined in cell-free systems for protein synthesis. No inhibition of protein synthesis was seen using intact, heat-treated and alkaline-treated virus RNA. Added virus RNA stimulated amino acid incorporation in cell-free systems from E. coli and chick cells. This is used as an evidence that high molecular weight virus RNA may serve as a template for protein synthesis in vivo.
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