Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines.

J F Atkins,J B Lewis,C W Anderson,R F Gesteland

doi:10.1016/s0021-9258(19)41234-9

Abstract

Addition of the polyamines spermidine, spermine, or putrescine to a fractionated mammalian cell-free protein-synthesis system programmed by a variety of mRNAs results in a 3- to 5-fold stimulation of amino acid incorporation over that found in the absence of added polyamine. The mRNAs used as template were adenovirus mRNA, globin 9s mRNA, and RNA from the bacteriophages R17, Qbeta, and MS2. The relative amounts of 10 adenovirus polypeptides synthesized in vitro are altered by the addition of polyamines to the translation system to reflect more closely the relative amounts of these polypeptides synthesized in vivo. This qualititive improvement in translation products on addition of polyamines allow the analysis of a number of products which are at best only marginally synthesized in the absence of added polyamines. The low level of synthesis due to endogenous mRNA is stimulated by spermidine and spermine but a lesser extent by putrescine.

Highlights

MethodsThe three major polyamines,putrescine,spermidine,and sperm- The system used for cell-free protein synthesis was that of ine, are found in different relative amountsin different cells
With the natural mRNA from MS2 bacteriophage,the Mg2f could be partially replaced by polyaminesbut the over-all synthesiswasnot enhanced[12]
It hasbeenfound that spermineenhancestranslation of natural mRNAs in cell-free extracts from wheat germ’ [13], with a large enhancementof the synthesisof larger polypeptides.’

Summary

Methods

The three major polyamines,putrescine,spermidine,and sperm- The system used for cell-free protein synthesis was that of ine, are found in different relative amountsin different cells. Schreier and Staehelin [19] as modified by Anderson et al [16]. Their distribution in cell fractions varies, but no absolutespeci- For some experiments, initiation factors were prepared from Krebs ficity is demonstrable.This is undoubtedly complicatedby the II ascites cells instead of from rabbit reticulocytes. Circumstantialevidenceimplicatestheir involvement in protein synthesis.Polyaminesare found in isolatedribosomesb, ut tion at 0” width Mutants of Escherichiucoli have beenisolated that cannot synthesizeputrescine(l), and when putrescineis of reticulocyte ribosomal wash previously The preparation of (9 S) globin mRNA bacteriophage RNAs [22, 23] and mRNA used [16]. Mutants of Escherichiucoli have beenisolated that cannot synthesizeputrescine(l), and when putrescineis of reticulocyte ribosomal wash previously The preparation of (9 S) globin mRNA bacteriophage RNAs [22, 23] and mRNA used [16]. (al), R17, MS2, and QB from the cytoplasm of addedto starved cellsprotein synthesisis stimulated immedi- cells infected with adenovirus both late [16] and early [24] after ately [2]

Results

Discussion

Conclusion