DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.
Background: Assimilatory NADPH-sulfite reductase (SiR) is an essential metalloenzyme for sulfur metabolism made from two subunits. Results: We defined how the subunits of SiR assemble, with or without cofactors. Conclusion: One region of the metalloenzyme interacted either with its reductase partner when cofactors were formed or with itself when they were not. Significance: We propose a novel mechanism to regulate SiR assembly.
The central step in the assimilation of sulfur is a six-electron reduction of sulfite to sulfide, catalyzed by the oxidoreductase NADPH-dependent assimilatory sulfite reductase (SiR). SiR is composed of two subunits. One is a multidomain flavin binding reductase (SiRFP) and the other an iron-containing oxidase (SiRHP). Both enzymes are primarily globular, as expected from their functions as redox enzymes. Consequently, we know a fair amount about their structures but not how they assemble. Curiously, both structures have conspicuous regions that are structurally undefined, leaving questions about their functions and raising the possibility that they are critical in forming the larger complex. Here, we used ultraviolet-visible and circular dichroism spectroscopy, isothermal titration calorimetry, proteolytic sensitivity tests, electrospray ionization mass spectrometry, and activity assays to explore the effect of altering specific amino acids in SiRFP on their function in the holoenzyme complex. Additionally, we used computational analysis to predict the propensity for intrinsic disorder within both subunits and found that SiRHP's N-terminus is predicted to have properties associated with intrinsic disorder. Both proteins also contained internal regions with properties indicative of intrinsic disorder. We showed that SiRHP's N-terminal disordered region is critical for complex formation. Together with our analysis of SiRFP amino acid variants, we show how molecular interactions outside the core of each SiR globular enzyme drive complex assembly of this prototypical oxidoreductase.
Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the
Deltaproteobacteria
branch coordinates expression of two Fe-S cluster biogenesis machineries.
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