Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3’UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3’UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.DOI: http://dx.doi.org/10.7554/eLife.22883.001

How plus-strand [+]RNA virus genomes transition from translation templates to replication templates is a matter of much speculation. We have previously proposed that, for Turnip crinkle virus, binding of the encoded RNA-dependent RNA polymerase (RdRp) to the 3'UTR of the [+]RNA template promotes a regional wide-spread conformational switch to an alternative structure that disassembles the cap-independent translation enhancer (CITE) in the 3'UTR. The active 3'CITE folds into a tRNA-like T-shaped structure (TSS) that binds to 80S ribosomes and 60S subunits in the P-site. In this Point-of-View, we discuss the history of our research on the TSS and our recent report combining coarse level single molecule force spectroscopy (optical tweezers) with fine-grain computer simulations of this experimental process and biochemical approaches to obtain a detailed understanding of how RdRp binding in the TSS vicinity might lead to an extensive rearrangement of the RNA structure.

Ca imaging techniques to visualize the spread of the impulse across the entire network of SAN cells and local Ca release dynamics within individual cells. Immunolabeling of Connexin-43 and HCN4 was employed to correlate cytoarchitecture with Ca activity. Rhythmic AP-induced Ca transients (APCTs), which correlated with APs recorded simultaneously using sharp microelectrode, were employed to monitor the impulse conduction across the SAN. The site at which the earliest APCTs occurred, identified by phase shift analysis, was between crista terminalis and superior vena cava. The APCT travels from this area across the entire SAN along curvilinear bundles of striated SAN cells rich in Connexion-43 and weak in HCN4 immunolabeling. LCRs were observed in all areas of SAN, including cells within the conduction bundles. Cells close to the APCT initiation site generated only rhythmic LCRs (without APCTs) and these LCRs preceded APCTs in the cells of the APCT-initiation area. Cells close to APCT initiation site were rich in HCN4, but essentially devoid of Connexin-43 and striations. Our results are consistent with the hypothesis that a coupled-clock system operates both within and among cells comprising the SAN network: some specialized cells generate only rhythmic LCRs that ignite adjacent cells to initiate the impulse that propagates throughout the SAN cell network.

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3'UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/É 3 required five upstream adenylates, and H4a/É 3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg 2+ . SMD recapitulated the TSS unfolding order in the absence of Mg 2+ , showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3'UTR, suggesting that RdRp binding to the adenylates disrupts H4a/ É 3 , leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.