Effects of ions on mrna elements, ribosomes and antibiotic-RNA interactions - recent interest in mrna vaccines and mrna therapies have brought ribosome mechanism and mrna mechanism into focus

Despite decades of research on the mechanism of translation of mrna into protein and ribosome function, the underlying physics of ribosome operation are still not well understood at the atomistic level. Because IT is a simpler system, and because we are keenly interested in the biomedical problem of drug-resistant bacteria, we focus on the bacterial ribosome. Even for the bacterial ribosome, fundamental questions remain open for all stages of protein synthesis. Since 2002, my lab has focused on trna selection ('decoding') and the effects of antibiotics on this process. We have used large-scale molecular simulations to identify the accommodation corridor, elements of the ribosome important in trna selection, and some of the operation principles. However, many questions remain unanswered: 1. How does initial codon sampling enable quick recognition of correct trnas and rejection of incorrect trnas? 2. How, mechanistically, do ions affect this process, in terms of inner sphere, outer sphere and diffuse magnesium? 3. How do the ions affect antibiotic interactions? The advent of time-resolved cryo-EM studies of ribosomes, along with high resolution cryo-EM structures make this an exciting time for ribosome research. The time-resolved cryo-EM studies are highly synergistic with our molecular simulations. The high resolution structures offer new information about magnesium ions and enable US to study key issues about how ions impact ribosome function. My lab has 20 years of prior studies and 30 million CPU hours currently allocated at LOS alamos national laboratory, along with data from cryo-EM, microbiologist and single molecule fret experimentalist collaborators to answer these questions. A related, but even less well understood problem is understanding exactly how the properties of mrna itself allow IT to regulate transcription and translation. Since 2008, my lab has used both wetlab experimental biochemistry approaches and molecular simulation approaches to understand the operational principles of ligand-binding elements in the 5' untranslated region of mrna (riboswitch mrna elements). We find that magnesium ions and the ligand cooperate to control the switch operation of the riboswitch. We identified 3 classes of magnesium ions: (i) diffuse ions that form an overall cloud around the RNA, (II) chelated inner sphere ions that bind directly to the RNA, and (III) outer sphere ions that are hexahydrated. In light of the relatively recent discovery of a gram-negative antibiotic that targets the FMN riboswitch, we will use enhanced sampling explicit solvent molecular dynamics simulations and coarse-grain explicit ION molecular simulations of riboswitches to address the fundamental questions: 1. What are the relative roles of inner and outer sphere magnesium ions in riboswitch operation? 2. How does the antibiotic affect these processes to alter regulation of transcription? We will use a variety of molecular simulation techniques and enhanced sampling techniques, in combination with cryo-EM, saxs and smfret data to disentangle the 3 ION effects to obtain a comprehensive picture of the dynamics of ION interactions and how they work together with antibiotic molecules to regulate transcription and translation. We will strive to recruit and retain a highly diverse team and participate in outreach activities to our local community.