High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

Abstract

The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins, but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends upon ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the C-terminal extension results primarily from a steric block. Finally, we identified two conserved regions, one the previously noted post-II region in the helicase core and the other in the C-terminal extension, which may help displace or sequester the opposite RNA strand during RNA unwinding.

An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

“…While sequence conservation of this loop is high within PriA homologues and partially conserved with the RecQ ARL, any conservation is not clear across all SF2 enzymes. However, elements directly following motif II, called ‘motif IIa’ in several other SF2 helicases, have identified residues that appear to be important for their helicase functions (Supplementary Figure S1B) (13,15–21). Given the similarities between the PriA ARL and RecQ ARL, we hypothesized that the PriA ARL could provide a critical element for coupling DNA binding to ATPase in the enzyme.…”

“…This segment in RecQ DNA helicases was termed an aromatic-rich loop (ARL) or motif IIa, and it has been shown to couple DNA-binding with ATP hydrolysis (13,16,17). Similarly positioned segments have been implicated in nucleic acid binding and/or ATPase activities in a small number of other SF2 subfamily helicases (18–21), but whether ARL/motif IIa elements are generally involved in the helicase mechanisms of other SF2 enzymes is not known.…”

Exploring the Molecular Basis of dsRNA Recognition by Mss116p Using Molecular Dynamics Simulations and Free-Energy Calculations

“…Besides, some studies also indicate that mutations of residues such as Q412A and D441A would influence the unwinding activity. 18,19 Why and how do the mutants reduce the dsRNA binding? So far, little is known about the essence of dsRNA binding, which is the initial step in the whole unwinding process.…”

Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs

“…1). The core contains 13 conserved sequence motifs, many of which are implicated in specific steps of substrate binding and RNA duplex unwinding (11). In most DEAD-box proteins, the core is flanked by additional N- and/or C-terminal extensions, which contribute to the functional diversity of this protein family.…”