Translational frame shifting in theEscherichia colidnaX genein vitro

Abstract

Production of the γ subunit of Escherichia coli DNA polymerase III holoenzyme is dependent on a very efficient translational frameshift in the dnaX gene. Iused an E.coli in vitro translation system to analyze the mechanism of this frameshifting event. In this system, γ was produced almost to the same extent as the inframe translation product, τ, suggesting that efficient frameshifting was reproduced In vitro. Coupling with transcription was not necessary for frameshifting. Addition of purified τ or γ had no effect on the frameshifting process suggesting the absence of direct feedback regulation. By use of mutant genes, a strong pausing site was identified at or very close to the frameshift site. This pausing was apparently caused by a potential stem-loop structure which was previously shown to enhance frameshifting. Thus, enhancement of frameshifting by this putative stem-loop seems to be mediated by the translation pausing at the frameshift site. Despite the apparent structural similarity of the dnaX frameshift site to that of the eukaryotlc retroviral genes, dnaX mRNA synthesized in vitro failed to direct the production of γ in eukaryotic translation systems. This suggests that frameshifting In the dnaX gene depends on components specific to the E.coli translation system.