Real-time detection of human telomerase DNA synthesis by multiplexed single-molecule FRET.

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we report the development and proof-of-concept results of a single-molecule approach to monitor the catalytic activity of human telomerase in real time and with single-nucleotide resolution. Using zero-mode waveguides and multicolor FRET, we recorded the processive addition of multiple telomeric repeats to individual DNA primers. Unlike existing biophysical and biochemical tools, the novel approach enables the quantification of nucleotide-binding kinetics before nucleotide incorporation. Moreover, it provides a means to dissect the unique translocation dynamics that telomerase must undergo after synthesis of each hexameric DNA repeat. We observed an unexpectedly prolonged binding dwell time of dGTP in the enzyme active site at the start of each repeat synthesis cycle, suggesting that telomerase translocation is composed of multiple rate-contributing sub-steps that evade classical biochemical analysis.

Peptide exchange on MHC-I by TAPBPR is driven by a negative allostery release cycle.

Chaperones TAPBPR and tapasin associate with class I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, wherein the lifetime and structural features of transiently bound peptides control the regulation of a conformational switch located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex.