The ribosome performs the essential function of protein synthesis in all living cells. The major functional form of bacterial ribosomes is a 70S complex, which is composed of two subunits, 30S and 50S. The interactions between the two subunits are clustered into several regions of the subunit interface and referred to as intersubunit bridges. Bridge interactions form during the subunit-joining step of translation initiation, which is critical for establishing the open reading frame of translation, while disruption of bridges during ribosome recycling helps to return both subunits into the translating pool. For the translation machinery to move along the messenger RNA (mRNA), bridges undergo large-scale rearrangement during each cycle of translation elongation. Therefore, the structure and dynamics of intersubunit bridges are crucial for ribosomal functions.
In this study, we investigate the molecular mechanism by which bridge interactions and interface dynamics contribute to translation. By targeting helix H69 of 23S rRNA and helix h44 of 16S rRNA, which together form bridge B2a/d, we elucidate the roles of this bridge in translation initiation (Chapter 2 and 3). Our data show that the competitive binding between H69 and IF3 and the conformational change of h44 in response to IF1 both contribute to the accuracy of start codon selection during subunit joining. Our findings suggest that the fidelity of translation initiation is largely governed by the intrinsic stability of codon-anticodon pairing in the 30S P site, while initiation factors kinetically regulate the process. Using a systematic approach, we identify bridge interactions contributing to the energy barrier of translocation (Chapter 4). Since these bridges (B1a, B4, B7a, and B8) are predicted to constrain head swiveling and intersubunit rotation, our date support a model in which both types of intersubunit movement are involved in formation of the transition state of translocation. Finally, we provide structural and functional evidence that cellular free 30S subunits are in an inactive state (Chapter 5), suggesting that conformational change at the 30S interface could serve as a functional switch for translation regulation.