The influence of downstream structured elements within mRNA on the dynamics of intersubunit rotation in ribosomes

Abstract

Proper codon/anticodon pairing within the ribosome necessitates linearity of the transcript. Any structures formed within a messenger RNA (mRNA) must be unwound before the respective codon is interpreted. Linearity, however, is not always the norm; some intricate structures within mRNA are able to exert unique ribosome/mRNA interactions to regulate translation. Intrinsic kinetic and thermal stability in many of these structures are efficient in slowing translation causing pausing of the ribosome. Altered translation kinetics arising from atypical interactions have been shown to affect intersubunit rotation. Here, we employ single-molecule Förster resonance energy transfer (smFRET) to observe changes in intersubunit rotation of the ribosome as it approaches downstream structured nucleic acid. The emergence of the hyperrotated state is critically dependent on the distance between downstream structure and the ribosome, suggesting interactions with the helicase center are allosterically coupled to intersubunit rotation. Further, molecular dynamics (MD) simulations were performed to determine ribosomal protein/mRNA interactions that may play a pivotal role in helicase activity and ultimately unwinding of downstream structure.