An mRNA Downstream Structure Promotes Frameshifting by Destabilizing the Hybrid State and Impeding the EF-G Driven Translocation Process

Abstract

Translational frameshifting occurs when a ribosome slips one or two nucleotides on a messenger RNA and generates a new sequence of amino acids. Many viral RNAs have programed frameshift-promoting signals to produce their proteins in the precise ratio needed for their viability. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1 programmed frameshifting by the dnaX gene in E. coli. The frameshifting mRNA has the usual three prokaryotic frameshifting signals: an internal Shine-Dalgarno sequence, a slippery sequence, and a stem loop. One round of translational elongation of the slippery sequence was characterized by the FRET changes between a Cy3-labeled L1 stalk in the 50S subunit and a Cy5-tRNALys in the P-site. We observed that the downstream stem loop, a critical signal for efficient frameshifting, destabilizes the hybrid state and thus shifts the equilibrium toward to the classical state of pre-translocation complexes. Translocation catalyzed by EF-G was significantly slower in the frameshifting mRNA than in the non-frameshifting mRNA lacking the stem loop. Furthermore, pre-translocation complexes of the frameshifting mRNA underwent several transitions between the classical and hybrid states in the presence of EF-G prior to complete translocation, while the majority of the non-frameshifting mRNA translocated rapidly via a single hybrid state. Quantitative analysis showed that the stem loop impedes EF-G driven translocation in the 30S subunit by elevating the activation barriers to translocation, and leaves the EF-G bound-hybrid state in dynamic equilibrium with the hybrid and classical states. We propose that by keeping the ribosome and tRNAs in the dynamically transiting pre-translocation states, the frameshifting mRNA allows more time for the ribosome to explore other paths, such as −1 frameshifting.