Origin of multiple intersubunit rotations before EF-G-catalyzed ribosomal translocation through the mRNA with a downstream secondary structure

Abstract

Background: A ribosome can translate through both the single-stranded region and duplex region of messenger RNA (mRNA). Recent single molecule fluorescence energy transfer (smFRET) data showed that when the ribosome translates through the mRNA containing a downstream stem loop, the ribosomal complex exhibits multiple fluctuations between the classical non-rotated and hybrid states before undergoing translocation in the presence of elongation factor G and GTP (EF-G.GTP) of high concentration; by contrast, when the ribosome translates through the mRNA lacking the stem loop, the complex samples the hybrid state approximately once before undergoing translocation. Results: Based on our proposed model, we provide theoretical analyses of the dynamics of the multiple fluctuations before undergoing mRNA translocation, providing quantitative explanations of the smFRET data. Conclusions: The good quantitative agreement between the theoretical data and the smFRET data supports the model showing that at saturating EF-G.GTP the multiple fluctuations with the mRNA containing the stem loop occur with EF-G still bound to the ribosome, rather than occur after the release of EF-G from the hybrid state and before the re-binding of EF-G; the multiple fluctuations are induced by occurrences of the futile transition during the mRNA translocation, which is induced by the resistance force resulting from the unwinding of the downstream mRNA stem loop to impede the ribosomal translocation; by contrast, with the mRNA lacking the stem loop, no resistance is present to impede the translocation and thus, no futile transition occurs, giving the ribosomal complex sampling the hybrid state once before undergoing translocation.