DNA sequesters endogenous mRNA during preparation of crude Escherichia coli extracts for protein synthesis; use of an S60 reduces the sequestered mRNA

Abstract

High backgrounds of endogenous incorporation of amino acids into protein in crude extracts of Escherichia coli are a consequence of endogenous messenger RNA. This RNA survives the preparation by virtue of protection or sequestering by endogenous DNA. Thus, DNase treatment in the crude extract leads to the elimination of this mRNA, while DNase treatment has no effect on the purified RNA. The endogenous mRNA also appears to be physically associated with DNA on CsCl gradients, and can be largely removed along with DNA by centrifugation of extracts at 60,000g. The top layer above a 60,000g centrifugation (S60) appears to be suitable for protein synthesis, and provides for lower background levels of endogenous messenger RNA.