DEAD-box proteins are ubiquitous in RNA metabolism and use ATP to mediate RNA conformational changes. These proteins have been suggested to use a fundamentally different mechanism from the related DNA and RNA helicases, generating local strand separation while remaining tethered through additional interactions with structured RNAs and RNA-protein (RNP) complexes. Here, we provide a critical test of this model by measuring the number of ATP molecules hydrolyzed by DEAD-box proteins as they separate short RNA helices characteristic of structured RNAs (6 -11 bp). We show that the DEAD-box protein CYT-19 can achieve complete strand separation using a single ATP, and that 2 related proteins, Mss116p and Ded1p, display similar behavior. Under some conditions, considerably <1 ATP is hydrolyzed per separation event, even though strand separation is strongly dependent on ATP and is not supported by the nucleotide analog AMP-PNP. Thus, ATP strongly enhances strand separation activity even without being hydrolyzed, most likely by eliciting or stabilizing a protein conformation that promotes strand separation, and AMP-PNP does not mimic ATP in this regard. Together, our results show that DEADbox proteins can disrupt short duplexes by using a single cycle of ATP-dependent conformational changes, strongly supporting and extending models in which DEAD-box proteins perform local rearrangements while remaining tethered to their target RNAs or RNP complexes. This mechanism may underlie the functions of DEAD-box proteins by allowing them to generate local rearrangements without disrupting the global structures of their targets.CYT-19 ͉ group I intron ͉ RNA chaperone ͉ RNA folding ͉ RNA helicase S tructured RNAs and RNA-protein complexes (RNPs) mediate a host of essential cellular processes, including processing of messenger RNAs and their translation into protein. In addition to folding into defined structures, many of these RNAs and RNPs undergo extensive conformational changes during their functions. Both their initial folding and conformational changes typically require DEAD-box proteins, which use ATP to promote RNA structural transitions.DEAD-box proteins are members of helicase superfamily-2 (SF2) and are related to the ATP-dependent RNA and DNA helicases that function in replication and other aspects of nucleic acid metabolism (1). However, rather than unwinding long, continuous duplexes, many DEAD-box proteins manipulate highly structured RNAs and RNPs by facilitating rearrangements that can include local disruptions of secondary structure, tertiary structure, and RNA-protein interactions.Consistent with their distinct functions, recent in vitro studies have strongly suggested that DEAD-box proteins operate on structured RNAs by a mechanism that is fundamentally different from processive helicases. The Neurospora crassa CYT-19 protein is required for proper folding of several mitochondrial group I introns in Neurospora crassa (2). It also assists folding of diverse group I and group II introns in vitro or when expressed in...

The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize non-native intronic structure. Nevertheless, Mss116p acceleration of all three constructs depends upon ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mtDNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing conversion of non-functional to functional RNA structure.

Single-molecule fluorescence experiments reveal how DEAD-box proteins unfold structured RNAs to promote conformational transitions and refolding to the native functional state.

The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility.

An interactive classroom demonstration that enhances students’ knowledge of steady-state and Michaelis–Menten enzyme kinetics is described. The instructor uses a free version of professional-quality KinTek Explorer simulation software and student input to construct dynamic versions of three static hallmark images commonly used to introduce enzyme kinetic concepts. The software, with its ability to change experimental conditions and immediately observe the effects on the kinetics of a system, allows students to be more aware of the experimental conditions that must exist for the assumptions of steady-state and Michaelis–Menten kinetics to be valid. Students report an increased understanding of the interplay between hallmark concentration versus time and rate versus concentration images. After the demonstration, students are prepared for data generation and analysis in a lab experiment focused on the steady-state. Additionally, they can begin independent simulation exercises using the software and are ready for discussions on pre-steady-state kinetics.