UC Berkeley

Escherichia coli ribosome engineering has emerged as a powerful approach to expand protein sequence-space to include monomers that are not α-amino acids. However, current ribosome engineering efforts are complicated by the enormous size and complexity of the ribosome—a 2.8 megadalton complex composed of two subunits (50S and 30S subunits), three ribosomal RNAs, and at least 54 ribosomal proteins. Peptide bond formation is catalyzed within the peptidyl transferase center (PTC), located within the 50S ribosomal subunit. The generation of a miniaturized PTC that recapitulates the native PTC fold and function will aid a rapid screening of ribosomal mutants that support new polymerization chemistry beyond the peptide bond. As a first step toward this effort, we seek to address a fundamental question: what is the smallest element of RNA capable of both binding acylated tRNA and catalyzing bond formation? The development of a minimal PTC that recapitulates many features of an intact ribosome is described in chapter 2 of this dissertation.In addition to the minimization approach, ribosome engineering efforts can be accelerated by the development of aminoacyl-tRNA synthetase (aaRS) variants that efficiently generate acyl-tRNA with non-α-amino acids in vivo. Traditional selection experiments to evolve aaRS mutants require ribosomal translation, making this selection system unsuitable if the desired non-L-α-amino acids are poorly tolerated by the ribosome. Therefore, a new selection strategy independent of ribosomal translation is required to ensure the rapid selection of new aaRS variants that recognize novel monomers. T-box riboswitches are RNA regulatory elements that sense tRNA acylation to regulate downstream gene expression in gram-positive bacteria. We envision that T-box riboswitches could be used to regulate gene expression in the selection of aaRS variants instead of ribosomal translation. Initial efforts toward T-box engineering are described in chapter 3 of this dissertation.