Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equilibrium binding measurements. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4.

Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are to promote rearrangements of structured RNAs and to remodel RNA-protein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. While all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms.

Highlights d A thermodynamic model quantitatively predicts PUM1/2 binding to any RNA sequence d Factors beyond simple recognition of consecutive residues influence binding d Comparison to X-linking data reveals thermodynamic control of PUM2 binding in cells d Analysis of RNA structure effects suggests disruption of RNA structure in cells

DEAD-box proteins are ubiquitous in RNA-mediated processes and function by coupling cycles of ATP binding and hydrolysis to changes in affinity for single-stranded RNA. Many DEAD-box proteins use this basic mechanism as the foundation for a version of RNA helicase activity, efficiently separating the strands of short RNA duplexes in a process that involves little or no translocation. This activity, coupled with mechanisms to direct different DEAD-box proteins to their physiological substrates, allows them to promote RNA folding steps and rearrangements and to accelerate remodeling of RNA-protein complexes. This review will describe the properties of DEAD-box proteins as RNA helicases and the current understanding of how the energy from ATPase activity is used to drive the separation of RNA duplex strands. It will then describe how the basic biochemical properties allow some DEAD-box proteins to function as chaperones by promoting RNA folding reactions, with a focus on the self-splicing group I and group II intron RNAs. KeywordsATPase; CYT-19; DExD/H-box protein; Group I intron; Group II intron; Mss116; RNA folding DEAD-box proteins are the largest family of superfamily 2 (SF2) helicases. They share with other SF2 proteins a helicase core composed of two RecA-like domains and a common set of sequence motifs. In the DEAD-box proteins, motif II includes the sequence D-E-A-D, which is the origin of their name (1). DEAD-box proteins are found in all three domains of life and perform a diverse set of functions in an even more diverse set of cellular processes, including assembly and function of macromolecular machines like the ribosome and spliceosome, quality control mechanisms in gene expression, nuclear export of mRNA, folding of self-splicing RNA introns, and others (2,3) ( Table 1). The broader term "DExD/ H-box" has been used to include other proteins with related motif II sequences (2,3), but a recent comparative sequence analysis has led to questions as to the relevance of this designation, because DEAD-box proteins are not more closely related to DExH helicases than to other families of SF2 helicases (4).In keeping with the broad range of functions for DEAD-box proteins, there is also considerable diversity in their mechanisms. Nevertheless, common themes are emerging. Since their discovery more than 20 years ago (1), the central defining biochemical activity of DEAD-box proteins has been their ability to bind and hydrolyze ATP in a cycle that is stimulated by binding to single-stranded or double-stranded RNA (ssRNA or dsRNA). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptSubsequent work has shown that the energetic communication implied by the stimulation of ATPase activity by RNA binding forms a common set of properties that underlies the diverse mechanisms of DEAD-box proteins. Progression through the ATPase cycle leads to changes in ssRNA affinity, allowing DEAD-box proteins to bind and release RNA in a regulated manner. This regulated binding is then used by different ...

The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.EAD-box proteins comprise the largest family of helicases and play critical roles in all aspects of RNA metabolism, including RNA splicing, translation, ribosome assembly, RNA degradation, and RNA transport (1, 2). Although their functions vary broadly, all DEAD-box proteins have a conserved helicase core consisting of two RecA-like domains (denoted domains 1 and 2) separated by a flexible linker and operate by a mechanism involving conformational changes within this core (3-6). These conformational changes couple cycles of ATP binding and hydrolysis to RNA binding and release by the core, enabling DEADbox proteins to promote local RNA unwinding and remodeling of structured RNAs and RNA-protein complexes (3-5).Structural and biochemical studies have given important insights into how DEAD-box proteins unwind RNA (2-4). The two helicase core domains are separated from each other in an open state in the absence of substrates, but they interact to form a compact, closed state upon binding of ATP and RNA. In this closed state, the interface between the two core domains forms a catalytic site for ATP hydrolysis and an RNA-binding cleft, which can accommodate a short region of a duplex strand (7). The tight binding of the RNA strand within this cleft results in a bend that is incompatible with partner-strand base pairing, leading to local RNA unwinding (7-10). ATP hydrolysis and dissociation of the P i product are then necessary to release the bound single-stranded RNA (9,11,12).Whereas the helicase core is central to the biochemical activities of DEAD-box proteins, most also have substantial extensions or additional domains at their N-and/or C-termini (1, 3). These extensions vary widely in size, composition, and function, but many are thought to interact with RNA or protein components of complexes to direct the DEAD-box proteins to desired sites of action. A common type of extension is a basic C-terminal sequence (C-tail), which is typically predicted...