ABSTRACT
It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.
Subject(s)
Codon , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Escherichia coli/virology , Macrocyclic Compounds/metabolism , Polymers/metabolism , Protein Biosynthesis , T-Phages/growth & development , Amino Acids/metabolism , Bacteriolysis , Codon Usage , Codon, Terminator , Directed Molecular Evolution , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Gene Deletion , Genetic Code , Genome, Bacterial , Macrocyclic Compounds/chemistry , Mutagenesis , Peptide Termination Factors/genetics , Polymers/chemistry , RNA, Bacterial/genetics , RNA, Transfer/genetics , RNA, Transfer, Ser/genetics , Ubiquitin/biosynthesis , Ubiquitin/geneticsABSTRACT
An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant.
