2 research outputs found
d‑Amino Acid-Mediated Translation Arrest Is Modulated by the Identity of the Incoming Aminoacyl-tRNA
A complete understanding
of the determinants that restrict d-amino acid incorporation
by the ribosome, which is of interest
to both basic biologists and the protein engineering community, remains
elusive. Previously, we demonstrated that d-amino acids are
successfully incorporated into the C-terminus of the nascent polypeptide
chain. Ribosomes carrying the resulting peptidyl-d-aminoacyl-tRNA
(peptidyl-d-aa-tRNA) donor substrate, however, partition
into subpopulations that either undergo translation arrest through
inactivation of the ribosomal peptidyl-transferase center (PTC) or
remain translationally competent. The proportion of each subpopulation
is determined by the identity of the d-amino acid side chain.
Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA)
acceptor substrate that is delivered to ribosomes carrying a peptidyl-d-aa-tRNA donor further modulates this partitioning. Our discovery
demonstrates that it is the pairing of the peptidyl-d-aa-tRNA
donor and the aa-tRNA acceptor that determines the activity of the
PTC. Moreover, we provide evidence that both the amino acid and tRNA
components of the aa-tRNA acceptor contribute synergistically
to the extent of arrest. The results of this work deepen our understanding
of the mechanism of d-amino acid-mediated translation arrest
and how cells avoid this precarious obstacle, reveal similarities
to other translation arrest mechanisms involving the PTC, and provide
a new route for improving the yields of engineered proteins containing d-amino acids
Single-Molecule Reaction Chemistry in Patterned Nanowells
A new approach to
synthetic chemistry is performed in ultraminiaturized, nanofabricated
reaction chambers. Using lithographically defined nanowells, we achieve
single-point covalent chemistry on hundreds of individual carbon nanotube
transistors, providing robust statistics and unprecedented spatial
resolution in adduct position. Each device acts as a sensor to detect,
in real-time and through quantized changes in conductance, single-point
functionalization of the nanotube as well as consecutive chemical
reactions, molecular interactions, and molecular conformational changes
occurring on the resulting single-molecule probe. In particular, we
use a set of sequential bioconjugation reactions to tether a single-strand
of DNA to the device and record its repeated, reversible folding into
a G-quadruplex structure. The stable covalent tether allows us to
measure the same molecule in different solutions, revealing the characteristic
increased stability of the G-quadruplex structure in the presence
of potassium ions (K<sup>+</sup>) versus sodium ions (Na<sup>+</sup>). Nanowell-confined reaction chemistry on carbon nanotube devices
offers a versatile method to isolate and monitor individual molecules
during successive chemical reactions over an extended period of time