Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

Abstract

We explore the interactions of CYT-19, a DExD͞H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD͞H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.group I RNA ͉ RNA folding ͉ RNA unwinding ͉ Tetrahymena ribozyme E ssentially all cellular processes that are mediated by structured RNAs also require one or more DExD͞H-box proteins (1). These proteins use the energy from ATP binding and hydrolysis to accelerate RNA structural transitions, which can represent folding steps toward the native state or conformational switches between functional forms. The requirement for proteins presumably arises because RNA base pairs and other local structure can be highly stable even in the absence of enforcing structure, such that folding steps or rearrangements that require significant unfolding require assistance to proceed efficiently (2-4).Despite their ubiquitous presence, key questions about the functions of DExD͞H-box proteins remain largely unanswered. First, what interactions direct different DExD͞H-box proteins to their physiological substrates? All of these proteins share a core ''helicase'' domain containing a set of conserved motifs, and most have additional domains, a few of which have been shown to recognize substrate RNAs or RNA-protein complexes (reviewed in ref. 5). On this basis, targeting has been proposed as a general role for these domains, and the specific interactions that target one DExD͞H-box protein have been delineated (6-9). Nevertheless, in general, the interactions that direct DExD͞H box proteins to their substrates remain to be identified.Second, h...

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

“…This curious result immediately suggested a physical model for the chaperone activity of CYT-19 and other DEAD-box proteins. Chaperones function by first binding nonspecifically to structured RNA, then rearranging RNA structure by unwinding double-stranded regions and perhaps other accessible structures [36]. This general model was also supported by a clever experiment in which the unwinding activity of Ded1p was shown to be enhanced by a ssRNA tethered through a streptavidin linker to a model RNA duplex [44].…”

“…Further, Mss116p and CYT-19 were shown to promote group I and group II intron splicing in vitro, as was the cytosolic DEAD-box protein, Ded1p [37][38][39]. Each of these proteins was also shown to unwind RNA helices non-specifically in an ATP-dependent manner [36,37,[39][40][41].…”

“…Furthermore, functional RNAs often require conformational transitions other than simple helical rearrangements. Since DEAD-box proteins may function differently from canonical DNA helicases, they have been less-judgmentally referred to as RNA-dependent ATPases or RNA "unwindases" [34][35][36].…”

“…In general, the accessibility of the targeted structural element determines CYT-19's unwinding efficiency. Reprinted from [36]. Copyright A schematic of the hierarchy of forces that determine RNA folding.…”

From unwinding to clamping — the DEAD box RNA helicase family

“…It has become clear that this is because DEAD box proteins do not unwind duplexes based on translocation on the RNA 1,6,28,30,31,33 . Instead, DEAD box proteins load directly onto the duplex region and then pry the strands apart in an ATP-dependent fashion 30,34 .…”