Functional circularity of legitimate Qbeta replicase templates

Qβ replicase (RNA-directed RNA polymerase of bacteriophage Qβ) exponentially amplifies certain RNAs in vitro. Previous studies have shown that Qβ replicase can initiate and elongate on a variety of RNAs; however, only a minute fraction of them are recognized as ‘legitimate’ templates. Guanosine 5′-triphosphate (GTP)-dependent initiation on a legitimate template generates a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid, a powerful inhibitor of RNA-protein interactions. On the contrary, initiation on an illegitimate template is GTP independent and does not result in the aurintricarboxylic-acid-resistant replicative complex. This article demonstrates that the 3′ and 5′ termini of a legitimate template cooperate during and after the initiation step. Breach of the cooperation by dividing the template into fragments or by introducing point mutations at the 5′ terminus reduces the rate and the yield of initiation, increases the GTP requirement, decreases the overall rate of template copying, and destabilizes the postinitiation replicative complex. These results revive the old idea of a functional circularity of legitimate Qβ replicase templates and complement the increasing body of evidence that functional circularity may be a common property of RNA templates directing the synthesis of either RNA or protein molecules.     

Non-Canonical Allostery in Cyclic Nucleotide Dependent Kinases

The cAMP- and cGMP-dependent protein kinases (PKA and PKG) are canonically activated by the corresponding cyclic nucleotides. However, both systems are also sensitive to a wide range of non-canonical allosteric effectors, such as reactive oxygen species, which induce the formation of regulatory inter- and intra-molecular disulfide bridges, and disease-related mutations (DRMs). Here, we present a combined analysis of representative non-canonical allosteric effectors for PKA and PKG, and we identify common molecular mechanisms underlying non-canonical allostery in these kinases, from shifts in dynamical regulatory equilibria to modulation of inter-protomer interactions. In addition, mutations may also drive oligomerization beyond dimerization, and possibly phase transitions, causing loss of kinase inhibitory function and amplifying the allosteric effects of DRMs. Hence non-canonical allosteric stimuli often result in constitutive kinase activation underlying either physiological control of downstream signaling pathways or pathological outcomes, from aortic aneurisms to cancer predisposition. Overall, PKA and PKG emerge as “pan-sensors” going well beyond canonical cyclic nucleotide activation, revealing their versatile roles as central signaling hubs.