In all organisms, proteins are synthesized by ribosomes, large RNA-based enzymes that use aminoacyl- tRNA substrates to translate messenger RNA. In recent years, tremendous progress has been made in elucidating the structure of the ribosome in the absence and presence of substrates and various translation factors. Despite this wealth of structural information, several important questions about the molecular mechanisms of translation remain open. Furthermore, there exist highly conserved protein factors known to interact with the ribosome whose roles in the cell remain unclear. The long-term goal of my laboratory is to fill these gaps in knowledge.
Aim 1. Over the past several years, we have been studying mutations in the RNA component (16S rRNA) of the small (30S) subunit that promote miscoding. We found that mutation G299A on the solvent side of the 30S subunit acts by disrupting intersubunit bridge B8, which lies 80 angstroms away. Experiments of Aim 1 will determine which other fidelity mutations (ribosomal ambiguity or ram, error-promoting; restrictive or res, error-reducing) act by allosterically altering B8 and whether codon-anticodon interaction is coupled to B8 disruption. This work will shed light on the conformational dynamics of the subunit that govern the decoding process.
Aim 2. In all cells, ribosome assembly / maturation is monitored by quality control mechanisms. These mechanisms are complex and remain incompletely understood. Our recent work on the conserved translational GTPase LepA suggests that the factor contributes to translation initiation, either directly or indirectly. We hypothesize that LepA plays a role in ribosome assembly / maturation, and subunits formed in its absence are functional but defective in initiation. Experiments of Aim 2 will investigate this hypothesis, and may elucidate the physiological role of LepA. Ribosomes are a main target of antibiotics, and defects in translation are associated with a growing number of inherited human diseases and cancers. Insight gained by this project may lead to the development of novel antimicrobial drugs and/or treatments for one or more hereditary diseases.
One of the largest challenges facing modern medicine is the emergence of antibiotic resistance, and many classes of medically useful antibiotics target the ribosome. Defects in protein synthesis have been attributed to many inherited human diseases, and a growing body of evidence suggests that alteration in ribosome biogenesis and/or activity plays an important role in the development of several cancers. Insight gained from this project may (1) aid efforts to develop novel antibiotics and/or therapy regimes to combat pathogens with multiple-drug resistance and (2) contribute to the treatment and/or prevention of one or more hereditary diseases and/or cancers.
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