Dec 23, 1980

Polysome-dependent in vitro translation system capable of peptide chain reinitiation

Biochemistry
V A AroskarN G Avadhani

Abstract

A sensitive in vitro translation system has been developed which makes use of cellular polysomes as the source of mRNA and ribosomes. The soluble factors are derived from the preincubated S-30 fraction by centrifugation through a discontinuous sucrose gradient. Of the four fractions tested, fraction 1 (topmost fraction in the gradient) and fraction 2 (fraction sedimenting in 0.5 M sucrose) were stimulatory. These two fractions together yield the highest activity, corresponding to about 125 times the background incorporation. The polysome-directed system exhibits optimal activity in the range 1.8-2 mM Mg2+ and 125-175 mM KCl. The polysome-directed in vitro products exhibit a complexity comparable to the in vivo products resolved on the two-dimensional polyacrylamide gels of O'Farrell [O'Farrell, P. (1975) J. Biol. Chem. 250, 4007-4021]. The system is capable of active chain reinitiation as indicated by partial inhibition by 7-methylguanosine 5'-monophosphate and pactomycin and N-terminal end analysis of in vitro products. This system can also translate polysomes from diverse tissues such as mouse liver, rat liver, and rat brain. The levels and also the authenticity of translation of rat liver albumin and mouse liver carbamoyl ph...Continue Reading

Mentioned in this Paper

Polyribosomes
Alb
7-methylguanosine
Set of Polysomal Ribosomes
Polyacrylamide Gels
Protein Biosynthesis
Centrifugation
Soluble
Ribosomes
Alb

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