Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

Over the past two decades, one of the standard models of protein folding has been the "two-state" model, in which a protein only resides in the folded or fully unfolded states with a single pathway between them. Recent advances in spatial and temporal resolution of biophysical measurements have revealed "beyond-two-state" complexity in protein folding, even for small, single-domain proteins. In this work, we used high-resolution optical tweezers to investigate the folding/unfolding kinetics of the B1 domain of immunoglobulin-binding protein G (GB1), a well-studied model system. Experiments were performed for GB1 both in and out of equilibrium using force spectroscopy. When the force was gradually ramped, simple single-peak folding force distributions were observed, while multiple rupture peaks were seen in the unfolding force distributions, consistent with multiple force-dependent parallel unfolding pathways. Force-dependent folding and unfolding rate constants were directly determined by both force-jump and fixed-trap measurements. Monte Carlo modeling using these rate constants was in good agreement with the force ramp data. The unfolding rate constants exhibited two different behaviors at low vs high force. At high force, the unfolding rate constant increased with increasing force, as previously reported by high force, high pulling speed force ramp measurements. However, at low force, the situation reversed and the unfolding rate constant decreased with increasing force. Taken together, these data indicate that this small protein has multiple distinct pathways to the native state on the free energy landscape.

Telomerase | Telomere | Optical Tweezers | Single-Molecule Fluorescence | G-quadruplex | Cancer | AgingCorrespondence:schmi706@msu.edu (Twitter: @jenscs83) and mjcomsto@msu.eduTelomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells 1 . Telomerase is a reverse transcriptase composed of telomerase reverse transcriptase (TERT) 2 , telomerase RNA (TR) 3 , and several accessory proteins 4 . To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to the singlestranded overhangs at chromosome ends by copying the template region within its RNA subunit 5,6 . Between repeat additions, the RNA template must be recycled, which requires disrupting the base-pairing between TR and the substrate DNA 6 . How telomerase remains associated with the substrate DNA during this critical translocation step remains unknown. Here, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. Using a newly developed single molecule telomerase activity assay utilizing high-resolution optical tweezers, we directly measured stepwise, processive telomerase activity. We found that telomerase tightly associates with its DNA substrate, synthesizing multiple telomeric repeats before releasing them in a single large step. The rate at which product is released from the anchor site closely corresponds to the overall rate of product dissociation from elongating telomerase 7 , suggesting that it is a key parameter controlling telomerase processivity. We observed folding of the released product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into processive telomerase catalysis, a process critical for telomere length maintenance and therefore cancer cell survival 8,9 .To investigate substrate extension by a single telomerase ribonucleoprotein (RNP), we developed a single molecule telomerase activity assay using dual-trap high-resolution optical tweezers (Fig. 1a). Telomerase and its substrate were attached to separate polystyrene beads (Fig. 1b). The connection between the two beads was formed by the association of telomerase with its substrate DNA ( Fig. 1a,b). When applying a low constant force (4.0-4.5 pN) to the tether, substrate elongation by telomerase was measured as an increase in dis-tance between the two beads ( Fig. 1a). To attach telomerase to the bead, we utilized a 3xFLAG-HaloTag on TERT, modified with biotin ( Fig. 1b,c). This tag did not affect telomerase assembly, catalytic activity, processivity, or stimulation by POT1/TPP1 ( Fig. 1c,d, Extended Data Fig. 1a-c), indicating that it is fully functional.Using this experimental approach, we set out to analyze how telomerase processively synthesizes telomeric repeats. If substrate DNA is only bound to telomerase by basepairing to TR we would expect to observe a sequence of single nucleotide (nt) addition events as a result o...

solution measurements, and the challenges in incorporating them into computational prediction. As improvement opportunities were only partly realized in CASP12, we provide guidance on how data from the full-length biological unit and the solution state can better aid prediction of the folded monomer or subunit. We furthermore describe strategic integrations of solution measurements with computational prediction programs with the aim of substantially improving foundational knowledge and the accuracy of computational algorithms for biologically-relevant structure predictions for proteins in solution. 2856-Pos Board B64Mimicking Microbial Rhodopsin Isomerization Photoreceptor proteins, like microbial rhodopsins, play crucial roles in sensing and responding to light. The microbial rhodopsins all contain a retinylidene chromophore bound via Schiff base to a lysine. 1 Upon absorption of light, retinal isomerizes from all-trans to 13-cis, driving a protein conformational change; then the all-trans retinal is rapidly and thermally regenerated at the end of the photocycle. The rhodopsin must: channel the photo-isomerization to the C13-C14 double bond exclusively; overcome the energy barrier for photo-isomerization; and induce the rapid, thermal return to its original all trans form. 1,2 Research investigating this isomerization pathway has involved either chemical modification or mutational studies. An alternative approach would be to engineer a system from scratch that recapitulates all of the functions of a microbial rhodopsin, which would result in a much deeper understanding of the protein/chromophore interactions required to make a rhodopsin a reality. Herein we report just such a system using human Cellular Retinoic Acid Binding Protein II (hCRABPII) as a template. In this system, the photo-isomerization of retinal from all-trans to 13-cis 15-syn was demonstrated both in solution and in protein crystals, where atomic resolution structures after photo-isomerization showed both quantitative and specific photo-isomerization of the 13-bond and subsequent thermal relaxation exclusively to the all-trans isomer, fully recapitulating the microbial rhodopsin photocycle. In addition, the crystal structure of a 13-cis retinal-bound protein complex was determined at atomic resolution for the first time, showing how proteins can stabilize this isomeric form of retinal. 1-Ernst, O. et al.