Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes

Five different coding sequences of bacterial or eukaryotic origin in plasmi ds under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-lengt h product only in four of the five coding sequences tested. A unique patter n of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivativ e, cytidylic acid-puromycin, which was radioactively labeled. Thus these in complete polypeptides can be defined as nascent peptides bound to the ribos omal P site. Certain nascent peptides could be shifted into full-length pro tein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ o r reticulocyte cell-free system resulted in a 80-90% full-length product wi th no evidence for nascent polypeptides and translational pausing. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Scienc e B.V. All rights reserved.