The RNA helicase UPF1 is a key component of the nonsense mediated mRNA decay (NMD) pathway. Previous X-ray crystal structures of UPF1 elucidated the molecular mechanisms of its catalytic activity and regulation. In this study, we examine features of the UPF1 core and identify a structural element that adopts different conformations in the various nucleotide- and RNA-bound states of UPF1. We demonstrate, using biochemical and single molecule assays, that this structural element modulates UPF1 catalytic activity and thereby refer to it as the regulatory loop. Interestingly, there are two alternatively spliced isoforms of UPF1 in mammals which differ only in the lengths of their regulatory loops. The loop in isoform 1 (UPF11) is 11 residues longer than that of isoform 2. We find that this small insertion in UPF11 leads to a two-fold increase in its translocation and ATPase activities. To determine the mechanistic basis of this differential catalytic activity, we have determined the X-ray crystal structure of the helicase core of UPF11 in its apo-state. Our results point toward a novel mechanism of regulation of RNA helicases, wherein alternative splicing leads to subtle structural rearrangements within the protein that are critical to modulate enzyme movements and catalytic activity.

The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3’-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD). This pathway also involves the RNA helicase UPF1, which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway. Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway. We demonstrate the involvement of UPF2, a core NMD factor and a known activator of UPF1, in SMD. UPF2 acts as an adaptor between Stau1 and UPF1, stimulates the catalytic activity of UPF1 and plays a central role in the formation of an SMD-competent mRNP. Our study elucidates the molecular mechanisms of SMD and points towards extensive cross-talk between UPF1-mediated mRNA decay pathways in cells.

The relative levels of different σ factors dictate the expression profile of a bacterium. Extracytoplasmic function σ factors synchronize the transcriptional profile with environmental conditions. The cellular concentration of free extracytoplasmic function σ factors is regulated by the localization of this protein in a σ/anti-σ complex. Anti-σ factors are multi-domain proteins with a receptor to sense environmental stimuli and a conserved anti-σ domain (ASD) that binds a σ factor. Here we describe the structure of Mycobacterium tuberculosis anti-σD (RsdA) in complex with the -35 promoter binding domain of σD (σD4). We note distinct conformational features that enable the release of σD by the selective proteolysis of the ASD in RsdA. The structural and biochemical features of the σD/RsdA complex provide a basis to reconcile diverse regulatory mechanisms that govern σ/anti-σ interactions despite high overall structural similarity. Multiple regulatory mechanisms embedded in an ASD scaffold thus provide an elegant route to rapidly re-engineer the expression profile of a bacterium in response to an environmental stimulus.

The Mycobacterium tuberculosis transcriptional regulator Rv1364c regulates the activity of the stress response σ factor σ F . This multi-domain protein has several components: a signaling PAS domain and an effector segment comprising of a phosphatase, a kinase and an anti-anti-σ factor domain. Based on Small Angle X-Ray Scattering (SAXS) data, Rv1364c was recently shown to be a homo-dimer and adopt an elongated conformation in solution. The PAS domain could not be modeled into the structural envelope due to poor sequence similarity with known PAS proteins. The crystal structure of the PAS domain described here provides a structural basis for the dimerization of Rv1364c. It thus appears likely that the PAS domain regulates the anti-σ activity of Rv1364c by oligomerization. A structural comparison with other characterized PAS domains reveal several sequence and conformational features that could facilitate ligand binding-a feature which suggests that the function of Rv1364c could potentially be governed by specific cellular signals or metabolic cues.