Alternate conformational trajectories in ribosome translocation.

Translocation in protein synthesis entails the efficient and accurate movement of the mRNA-[tRNA]2 substrate through the ribosome after peptide bond formation. An essential conformational change during this process is the swiveling of the small subunit head domain about two rRNA 'hinge' elements. Using iterative selection and molecular dynamics simulations, we derive alternate hinge elements capable of translocation in vitro and in vivo and describe their effects on the conformational trajectory of the EF-G-bound, translocating ribosome.

Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye.

Ribosome Pool Engineering Increases Protein Biosynthesis Yields.

The biosynthetic capability of the bacterial ribosome motivates efforts to understand and harness sequence-optimized versions for synthetic biology. However, functional differences between natively occurring ribosomal RNA (rRNA) operon sequences remain poorly characterized. Here, we use an ribosome synthesis and translation platform to measure protein production capabilities of ribosomes derived from all unique combinations of 16S and 23S rRNAs from seven distinct rRNA operon sequences.

Cell-free Biosynthesis of Peptidomimetics.

A wide variety of peptidomimetics (peptide analogs) possessing innovative biological functions have been brought forth as therapeutic candidates through cell-free protein synthesis (CFPS) systems. A key feature of these peptidomimetic drugs is the use of non-canonical amino acid building blocks with diverse biochemical properties that expand functional diversity. Here, we summarize recent technologies leveraging CFPS platforms to expand the reach of peptidomimetics drugs.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.