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 plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-length product only in four of the five coding sequences tested. A unique pattern of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing.
Ramachandiran
V
V
Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.
Kramer
G
G
Hardesty
B
B
D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011691:Puromycin / Q000737:chemistry / Q000494:pharmacology; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D011691:Puromycin