Induced fit of RNA on binding the L7Ae protein to the kink-turn motif

The kink-turn is a widespread motif in RNA consisting of a three-nucleotide bulge flanked on one side by consecutive A3G mismatches. Important examples are found in the ribosome, U4 RNA, and in snoRNAs involved in RNA modification. The motif is a common protein binding site, and the RNA has been found to adopt a tightly kinked conformation in crystal structures. However, in free solution there is a dynamic exchange between kinked and extended conformations, with the equilibrium driven toward the kinked form by the addition of metal ions. Here we used fluorescence resonance energy transfer (FRET) to show that the L7Ae protein of Archaeoglobus fulgidus binds to RNA containing a kink-turn with nanomolar affinity, and induces folding into the tightly kinked conformation even in the absence of metal ions. Thus this RNA may act as a relatively flexible hinge during RNA folding, until fixed into its ultimate kinked structure by the binding of L7 or related protein.     

The importance of G.A hydrogen bonding in the metal ion- and protein-induced folding of a kink turn RNA

The kink turn (K-turn) is a common motif in RNA structure, found in many RNA species important in translation, RNA modification and splicing, and the control of gene expression. In general the K-turn comprises a three nucleotide bulge followed by trans sugar-Hoogsteen G·A pairs. The RNA adopts a tightly kinked conformation, and is a common target for binding proteins, exemplified by the L7Ae family. We have measured the rates of association and dissociation for the binding of L7Ae to the Kt-7 kink turn, from which we calculate an affinity of K_D = 10 pM. This high affinity is consistent with the role of this binding as the first stage in the assembly of key functional nucleoproteins such as box C/D snoRNP. Kink-turn RNA undergoes a two-state transition between the kinked conformation, and a more extended structure, and folding into the kinked form is induced by divalent metal ions, or by binding of proteins of the L7Ae class. The K-turn provides an excellent, simple model for RNA folding, which can be dissected at the atomic level. We have analyzed the contributions of the hydrogen bonds that form the G·A pairs to the ion- and protein-induced folding of the K-turn. We find that all four hydrogen bonds are important to the stability of the kinked form of the RNA, and we can now define all the important hydrogen bonding interactions that stabilize the K-turn. The high affinity of L7Ae binding is coupled to the induced folding of the K-turn, allowing some sub-optimal variants to adopt the kinked geometry. However, in all such cases the affinity is lowered, and the results underline the importance of both G·A pairs to the stability of the K-turn.     

Structure of the K-turn U4 RNA: a combined NMR and SANS study

K-turn motifs are universal RNA structural elements providing a binding platform for proteins in several cellular contexts. Their characteristic is a sharp kink in the phosphate backbone that puts the two helical stems of the protein-bound RNA at an angle of 60°. However, to date no high-resolution structure of a naked K-turn motif is available. Here, we present the first structural investigation at atomic resolution of an unbound K-turn RNA (the spliceosomal U4-Kt RNA) by a combination of NMR and small-angle neutron scattering data. With this study, we wish to address the question whether the K-turn structural motif assumes the sharply kinked conformation in the absence of protein binders and divalent cations. Previous studies have addressed this question by fluorescence resonance energy transfer, biochemical assays and molecular dynamics simulations, suggesting that the K-turn RNAs exist in equilibrium between a kinked conformation, which is competent for protein binding, and a more extended conformation, with the population distribution depending on the concentration of divalent cations. Our data shows that the U4-Kt RNA predominantly assumes the more extended conformation in the absence of proteins and divalent cations. The internal loop region is well structured but adopts a different conformation from the one observed in complex with proteins. Our data suggests that the K-turn consensus sequence does not per se code for the kinked conformation; instead the sharp backbone kink requires to be stabilized by protein binders.     

Elbow Flexibility of the kt38 RNA Kink-Turn Motif Investigated by Free-Energy Molecular Dynamics Simulations

Kink-turns (K-turns) are common structural motifs that can introduce sharp kinks into double-stranded RNA, and have been proposed to mediate large-scale motions in the ribosome. K-turns consist of a bulge loop region flanked by trans sugar-Hoogsteen G:A pairs, and the sharp kink conformation is stabilized by A-minor interactions (adenine contacting a G:C basepair in the minor groove). Umbrella-sampling molecular dynamics simulations were used to disrupt an A-minor interaction in the ribosomal kt38 turn and to calculate the associated free-energy change. Coupling of umbrella sampling with replica exchanges between neighboring umbrella-sampling intervals could further improve the convergence of the free-energy calculations. The simulations revealed a coupled A-minor disruption and global opening of the K-turn motif, and allowed us to characterize several intermediate A-minor conformations. The calculated free-energy profile indicated a meta-stable, semi-open structure of slightly higher free energy (∼1 kcal mol⁻¹), separated by a small free-energy barrier (∼1.5 kcal mol⁻¹) from the closed (highly kinked) form. Both K-turn states are stabilized by distinct variants of the A-minor interaction. Further opening of the K-turn structure required significantly larger free-energy changes. The semi-open form had a reduced kink angle compatible with experimental data on K-turn solution structures, and opening was coupled to a continuous global unwinding of the K-turn motif. The range of free-energy changes associated with kt38 opening and unwinding are compatible with the idea that K-turns may facilitate biologically relevant motions during large-scale ribosome dynamics.     

Plasticity of the RNA kink turn structural motif

The kink turn (K-turn) is an RNA structural motif found in many biologically significant RNAs. While most examples of the K-turn have a similar fold, the crystal structure of the Azoarcus group I intron revealed a novel RNA conformation, a reverse kink turn bent in the direction opposite that of a consensus K-turn. The reverse K-turn is bent toward the major grooves rather than the minor grooves of the flanking helices, yet the sequence differs from the K-turn consensus by only a single nucleotide. Here we demonstrate that the reverse bend direction is not solely defined by internal sequence elements, but is instead affected by structural elements external to the K-turn. It bends toward the major groove under the direction of a tetraloop–tetraloop receptor. The ability of one sequence to form two distinct structures demonstrates the inherent plasticity of the K-turn sequence. Such plasticity suggests that the K-turn is not a primary element in RNA folding, but instead is shaped by other structural elements within the RNA or ribonucleoprotein assembly.     

Biochemical characterization of the kink-turn RNA motif

RNA, which acts as a medium for transmitting genetic information, plays a variety of roles in a cell. As with proteins, elucidation of the three- dimensional (3D) structures of RNAs is important for understanding their various roles. Determination of the atomic structures of crystallized ribosome has enabled the identification of previously unknown RNA structural motifs. The kink-turn (K-turn or GA) motif, which causes a sharp bend in an RNA double helix, was identified as one of these structural motifs. To biochemically characterize the K-turn, the motif was inserted into a hinge region of P4-P6 RNA, which is the most extensively studied self-folding RNA, and its properties were investigated. The stability and metal ion requirement of the constructs containing three different K-turn motifs were analyzed using native PAGE and dimethyl sulfate (DMS) modification. The formation of the sharp bending structure depends on the presence of divalent cation like Mg²⁺ or Ca²⁺, although its required concentration is different for each motif.     

The role of specific 2'-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA

The role of 2'-hydroxyl groups in stabilizing the tightly kinked geometry of the kink-turn (K-turn) has been investigated. Individual 2'-OH groups have been removed by chemical synthesis, and the kinking of the RNA has been studied by gel electrophoresis and fluorescence resonance energy transfer. The results have been analyzed by reference to a database of 11 different crystallographic structures of K-turns. The potential hydrogen bonds fall into several classes. The most important are those in the core of the turn and ribose-phosphate interactions around the bulge. Of these the single most important hydrogen bond is one donated from the 2'-OH of the 5' nucleotide of the bulge to the N1 of the adenine of the kink-proximal A*G pair. This is present in all known K-turn structures, and removal of the 2'-OH completely prevents metal ion-induced folding. Hydrogen bonds formed in the minor grooves of the helical stems are less important, and removal of the participating 2'-OH groups leads to reduced impairment of folding. These interactions are generally more polymorphic, and hydrogen bonds probably form where possible, as permitted by the global structure.     

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.     

RNA kink turns to the left and to the right

A helix-loop-helix within the group I intron has most of the canonical sequence elements of a kink turn (K-turn), yet it bends in the opposite direction. The reverse K-turn kinks toward the major rather than the minor grooves of the flanking helices. This suggests that there are two distinct subclasses of tertiary structures that a K-turn secondary structure can adopt. The final structure may be specified by external factors, such as protein binding or the tertiary structural context, rather than the intrinsic conformation of the RNA.     

RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn

The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G?A and A?G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make?a?long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure.     

Functional implications for a prototypical K-turn binding protein from structural and dynamical studies of 15.5K

The kink-turn (K-turn) motif is recognized and bound by a family of proteins that act as nucleation factors for ribonucleoparticle assembly. The binding of various proteins to a conserved RNA structural motif known as the K-turn has been shown to be an important component of regulation in the ribosome, in the spliceosome, and in RNA modification. 15.5K is a prototypical example of a K-turn binding protein, which has been shown to bind the 5'-U4 stem-loop of the spliceosome and the box C/D motif. We describe the solution NMR structure of free 15.5K, as well as studies of conformational flexibility from 15N NMR relaxation and H/D exchange experiments. The protein appears well-structured aside from conformational fluctuation in alpha3. Flexibility in fast time scale motions and the observation of limited intermediate and slow motions further characterize the free protein and may suggest local contributions to recognition and binding.     

Single-Molecule Observation of the Induction of k-Turn RNA Structure on Binding L7Ae Protein

The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form.     

The dynamic nature of the four-way junction of the hepatitis C virus IRES

Translation is initiated within the RNA of the hepatitis C virus at the internal ribosome entry site (IRES). The IRES is a 341-nucleotide element that contains a four-way helical junction (IIIabc) as a functionally important element of the secondary structure. The junction has three additional, nonpaired nucleotides at the point of strand exchange on one diagonal. We have studied the global conformation and folding of this junction in solution, using comparative gel electrophoresis and steady-state and time-resolved fluorescence resonance energy transfer. In the absence of divalent metal ions, the junction adopts an extended-square structure, in contrast to perfect four-way RNA junctions, which retain coaxial helical stacking under all conditions. The IIIabc junction is induced to fold on addition of Mg(2+), by pairwise coaxial stacking of arms, into the conformer in which the unpaired bases are located on the exchanging strands. Fluorescence lifetime measurements indicate that in the presence of Mg(2+) ions, the IIIabc junction exists in a dynamic equilibrium comprising approximately equal populations of antiparallel and parallel species. These dynamic properties may be important in mediating interactions between the IRES and the ribosome and initiation factors.     

Ribosomal RNA kink-turn motif--a flexible molecular hinge

Ribosomal RNA K-turn motifs are asymmetric internal loops characterized by a sharp bend in the phosphodiester backbone resulting in "V" shaped structures, recurrently observed in ribosomes and showing a high degree of sequence conservation. We have carried out extended explicit solvent molecular dynamics simulations of selected K-turns, in order to investigate their intrinsic structural and dynamical properties. The simulations reveal an unprecedented dynamical flexibility of the K-turns around their X-ray geometries. The K-turns sample, on the nanosecond timescale, different conformational substates. The overall behavior of the simulations suggests that the sampled geometries are essentially isoenergetic and separated by minimal energy barriers. The nanosecond dynamics of isolated K-turns can be qualitatively considered as motion of two rigid helix stems controlled by a very flexible internal loop which then leads to substantial hinge-like motions between the two stems. This internal dynamics of K-turns is strikingly different for example from the bacterial 5S rRNA Loop E motif or BWYV frameshifting pseudoknot which appear to be rigid in the same type of simulations. Bistability and flexibility of K-turns was also suggested by several recent biochemical studies. Although the results of MD simulations should be considered as a qualitative picture of the K-turn dynamics due to force field and sampling limitations, the main advantage of the MD technique is its ability to investigate the region close to K-turn ribosomal-like geometries. This part of the conformational space is not well characterized by the solution experiments due to large-scale conformational changes seen in the experiments. We suggest that K-turns are well suited to act as flexible structural elements of ribosomal RNA. They can for example be involved in mediation of large-scale motions or they can allow a smooth assembling of the other parts of the ribosome.     

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.     

Structure and folding of four putative kink turns identified in structured RNA species in a test of structural prediction rules

k-Turns are widespread key architectural elements that occur in many classes of RNA molecules. We have shown previously that their folding properties (whether or not they fold into their tightly kinked structure on addition of metal ions) and conformation depend on their local sequence, and we have elucidated a series of rules for prediction of these properties from sequence. In this work, we have expanded the rules for prediction of folding properties, and then applied the full set to predict the folding and conformation of four probable k-turns we have identified amongst 224 structured RNA species found in bacterial intergenenic regions by the Breaker lab (1). We have analyzed the ion-dependence of folding of the four k-turns using fluorescence resonance energy transfer, and determined the conformation of two of them using X-ray crystallography. We find that the experimental data fully conform to both the predicted folding and conformational properties. We conclude that our folding rules are robust, and can be applied to new k-turns of unknown characteristics with confidence.     

Inflammation in the avian spleen: timing is everything

Background

Human cells depend critically on the signal recognition particle (SRP) for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain.

Results

We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK) at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA.

Conclusions

The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.