Expression of different coding sequences in cell‐free bacterial and eukaryotic systems indicates translational pausing onEscherichia coliribosomes

Abstract

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 809 0% full-length product with no evidence for nascent polypeptides and translational pausing. ß

Experimental and Computational Analysis of Translation Products in Apomyoglobin Expression

“…The incompletely elongated polypeptides behave as on-path intermediates, in that they eventually convert into full-length protein as translation proceeds to completion. 53,63,77 Unlike the above pause site-related polypeptides, most of the incompletely translated apoMb species reported here are not converted to full-length protein upon extended incubation. Nearly all of the apoMb incomplete chains are maximally populated after 10-30 min and persist in solution even after 6 h of protein synthesis.…”

“…34,[52][53][54][55] In some cases, incomplete chains arise from transient ribosomal pausing followed by successful completion of elongation. This type of kinetic retardation is often related to either low codon usage 26,27,30 or mRNA local secondary/tertiary structure.…”

Evaluation of Approaches to Identify the Targets of Cellular Immunity on a Proteome-Wide Scale

“…For all four viral and one parasite antigens tested in this study, partial products were common. The production of partial fragments in E. coli based IVTT reactions has been described previously and attributed to a phenomena called translational pausing but the presence of partial products did not adversely affect immunogenicity or antigenicity [46]. Since the antigens produced by E. coli based IVTT were at least partially insoluble, simple techniques for purification, such as antibody coated beads, were not effective.…”

Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro

“…When CAT was synthesized in the E. coli cell‐free system, a distinct ladder of pause‐site peptides was observed in size from less than 3.0 kDa migrating close to the front of the gel to near full size of 25 kDa [1,2,17,18]. Accumulation of the smallest nascent peptide was pronounced, when protein synthesis was initiated with fluorophore‐[ 35 S]Met‐tRNA [2,17].…”