Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

Energetics of RNA binding by the West Nile virus RNA triphosphatase

The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5' end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.     

Deletion of a single hydrogen bonding atom from the MS2 RNA operator leads to dramatic rearrangements at the RNA-coat protein interface

The MS2 coat protein binds specifically to an RNA hairpin formed within the viral genome. By soaking different RNA fragments into crystals of MS2 coat protein capsids it is possible to determine the X-ray structure of the RNA–protein complexes formed. Here we present the structure to 2.85 Å resolution of a complex between a chemically modified RNA hairpin variant and the MS2 coat protein. This RNA variant has a substitution at the –5 base position, which has been shown previously to be pyrimidine-specific and is a uracil in the wild-type RNA. The modified RNA hairpin contains a pyridin-4-one base (4one) at this position that lacks the exocyclic 2-oxygen eliminating the possibility of forming a hydrogen bond to asparagine A87 in the protein. The 4one complex structure shows an unprecedented major conformational change in the loop region of the RNA, whereas there is almost no change in the conformation of the protein.     

Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA.

The structure of a ribonucleoprotein complex formed at the 5'-end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf-like structure and interact with both uncleaved 3CD, the viral protease-polymerase precursor, and a 36 kDa ribosome-associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem-loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site-directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic-protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5'-end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.     

RBPBind: Quantitative Prediction of Protein-RNA Interactions

There are hundreds of RNA binding proteins in the human genome alone and their interactions with messenger and other RNAs in a cell regulate every step in an RNA's life cycle. To understand this interplay of proteins and RNA it is important to be able to know which protein binds which RNA how strongly and where. Here, we introduce RBPBind, a web-based tool for the quantitative prediction of the interaction of single-stranded RNA binding proteins with target RNAs that fully takes into account the effect of RNA secondary structure on binding affinity. Given a user-specified RNA and a protein selected from a set of several RNA-binding proteins, RBPBind computes their binding curve and effective binding constant. The server also computes the probability that, at a given protein concentration, a protein molecule will bind to any particular nucleotide along the RNA. The sequence specificity of the protein-RNA interaction is parameterized from public RNAcompete experiments and integrated into the recursions of the Vienna RNA package to simultaneously take into account protein binding and RNA secondary structure. We validate our approach by comparison to experimentally determined binding affinities of the HuR protein for several RNAs of different sequence contexts from the literature, showing that integration of raw sequence affinities into RNA secondary structure prediction significantly improves the agreement between computationally predicted and experimentally measured binding affinities. Our resource thus provides a quick and easy way to obtain reliable predicted binding affinities and locations for single-stranded RNA binding proteins based on RNA sequence alone.     

Binding of U1A protein changes RNA dynamics as observed by 13C NMR relaxation studies

Recognition of RNA by proteins and small molecules often involves large changes in RNA structure and dynamics, yet very few studies have so far characterized these motional changes. Here we extend to the protein-bound RNA recent 13C relaxation studies of motions in the RNA recognized by human U1A protein, a well-known model for protein-RNA recognition. Changes in relaxation observed upon complex formation demonstrate that the protein-binding site becomes rigid in the complex, but the upper stem-loop that defines the secondary structure of this RNA experiences unexpected motional freedom. By using a helix elongation strategy, we observe that the upper stem-loop moves independently of the remainder of the structure also in the absence of U1A. Surprisingly, RNA residues making important intermolecular contacts in the structure of the complex exhibit increased flexibility in the presence of the protein. Both of these results support the hypothesis that RNA-binding proteins select a structure that optimizes intermolecular contacts in the manifold of conformations sampled by the free RNA and that protein binding quenches these motions. Together with previous studies of the RNA-bound protein, they also demonstrate that protein-RNA interfaces experience complex motions that modulate the strength of individual interactions.     

Functional domains of the RNA component of ribonuclease P revealed by chemical probing of mutant RNAs.

The higher-order structure of the RNA component of ribonuclease P from Escherichia coli was analyzed using chemical probes. The secondary structure model which had been constructed from the comparative sequence analysis of the RNA was refined using the experimental data. In a mutant RNA (A89 RNA), which contains a G----A substitution at nucleotide 89, we detected a number of conformational alterations clustered between nucleotides 90 and 239. In view of the fact that A89 RNA is as catalytically active as wild-type RNA, but defective in association with the protein component, it is clear that the catalytic function of the RNA component resides on the structure which is not disrupted by the A89 mutation and that the structures altered by the mutation represent the region(s) interacting with the protein component. Another mutant (A329 RNA), which has a G----A substitution at nucleotide 329 and is defective in catalytic function, showed no detectable change in higher-order structure.     

RNA binding property and RNA chaperone activity of dengue virus core protein and other viral RNA-interacting proteins

In this study we showed that the dengue virus (DENV) core protein forms a dimer with an α-helix-rich structure, binds RNA and facilitates the strand annealing process. To assess the RNA chaperone activity of this core protein and other dengue viral RNA-interacting proteins, such as NS3 helicase and NS5 proteins, we engineered cis- and trans-cleavage hammerhead ribozyme constructs carrying DENV genomic RNA elements. Our results indicate that DENV core protein facilitates typical hammerhead structure formation by acting as an RNA chaperone and DENV NS5 has a weak RNA chaperone activity, while DENV NS3 helicase failed to refold RNA with a complex secondary structure.     

Structure of RNA in satellite tobacco necrosis virus. A low resolution neutron diffraction study using 1H2O/2H2O solvent contrast variation

The crystal structure of satellite tobacco necrosis virus has been studied by neutron diffraction at 16 A resolution using the technique of 1H2O/2H2O solvent contrast variation to distinguish between the regions of protein and nucleic acid. The RNA density is essentially localized in a region just inside the protein coat, leading to a significant interaction between the two components. From the appearance of the RNA density we conclude that the protein coat imposes partial icosahedral symmetry on a significant proportion of the nucleic acid. The shape and dimensions of the major part of this density suggests that about 72% of the total RNA could be double-helical in structure. The most important interaction between the two components of the virus occurs between the N-terminal triple-helical arms of the protein subunits and those regions of the RNA density that could have a double-helical secondary structure.     

The crystal structure of a high affinity RNA stem-loop complexed with the bacteriophage MS2 capsid: further challenges in the modeling of ligand-RNA interactions

We have determined the structure to 2.8 A of an RNA aptamer (F5), containing 2'-deoxy-2-aminopurine (2AP) at the -10 position, complexed with MS2 coat protein by soaking the RNA into precrystallised MS2 capsids. The -10 position of the RNA is an important determinant of binding affinity for coat protein. Adenine at this position in other RNA stem-loops makes three hydrogen bonds to protein functional groups. Substituting 2AP for the -10 adenine in the F5 aptamer yields an RNA with the highest yet reported affinity for coat protein. The refined X-ray structure shows that the 2AP base makes an additional hydrogen bond to the protein compared to adenine that is presumably the principal origin of the increased affinity. There are also slight changes in phosphate backbone positions compared to unmodified F5 that probably also contribute to affinity. Such phosphate movements are common in structures of RNAs bound to the MS2 T = 3 protein shell and highlight problems for de novo design of RNA binding ligands.     

Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA.

Dimethyl sulfate modification of RNA in living Tetrahymena thermophila allowed assessment of RNA secondary structure and protein association. The self-splicing rRNA intron had the same methylation pattern in vivo as in vitro, indicating that the structures are equivalent and suggesting that this RNA is not stably associated with protein in the nucleolus. Methylation was consistent with the current secondary structure model. Much of telomerase RNA was protected from methylation in vivo, but the A's and C's in the template region were very reactive. Thus, most telomerase is not base paired to telomeres in vivo. Protein-free telomerase RNA adopts a structure different from that in vivo, especially in the template and pseudoknot regions. The U2 snRNA showed methylation protection at the Sm protein-binding sequence and the mRNA branch site recognition sequence. For both telomerase RNA and U2 snRNA, the in vivo methylation pattern corresponded much better to the structure determined by comparative sequence analysis than did the in vitro methylation pattern. Thus, as expected, comparative analysis gives the structure of the RNA in vivo.     

Isoenergetic microarrays to study the structure and interactions of DsrA and OxyS RNAs in two- and three-component complexes

Information on the secondary structure and interactions of RNA is important to understand the biological function of RNA as well as in applying RNA as a tool for therapeutic purposes. Recently, the isoenergetic microarray mapping method was developed to improve the prediction of RNA secondary structure. Herein, for the first time, isoenergetic microarrays were used to study the binding of RNA to protein or other RNAs as well as the interactions of two different RNAs and protein in a three-component complex. The RNAs used as models were the regulatory DsrA and OxyS RNAs from Escherichia coli, the fragments of their target mRNAs (fhlA and rpoS), and their complexes with Hfq protein. The collected results showed the advantages and some limitations of microarray mapping.     

A comparison of genotype-phenotype maps for RNA and proteins

The relationship between the genotype (sequence) and the phenotype (structure) of macromolecules affects their ability to evolve new structures and functions. We here compare the genotype space organization of proteins and RNA molecules to identify differences that may affect this ability. To this end, we computationally study the genotype-phenotype relationship for short RNA and lattice proteins of a reduced monomer alphabet size, to make exhaustive analysis and direct comparison of their genotype spaces feasible. We find that many fewer protein molecules than RNA molecules fold, but they fold into many more structures than RNA. In consequence, protein phenotypes have smaller genotype networks whose member genotypes tend to be more similar than for RNA phenotypes. Neighborhoods in sequence space of a given radius around an RNA molecule contain more novel structures than for protein molecules. We compare this property to evidence from natural RNA and protein molecules, and conclude that RNA genotype space may be more conducive to the evolution of new structure phenotypes.     

The antigen of hepatitis delta virus: examination of in vitro RNA-binding specificity.

The only known protein of hepatitis delta virus (HDV), the delta antigen, is found both within virus particles and within the nucleus of the infected cell, where it has one or more roles essential for RNA genome replication. Others have demonstrated that the antigen has the ability, in vitro, to specifically bind HDV RNA species. We report a further examination of this phenomenon, using partially purified recombinant protein, expressed as a fusion with the staphylococcal protein A. From Northwestern (RNA-immunoblot) analyses with both complete and various subdomains of HDV genomic and antigenomic RNAs, we found that a necessary feature for specific binding was that the RNA be able to fold to some extent into the so-called rodlike structure; this structure is a predicted intramolecular partial base-pairing of the circular RNA, with about 70% of all bases involved, so as to produce an unbranched rodlike structure. Six different subregions of the HDV rodlike structure, three on the genomic RNA and three on its complement, the antigenomic RNA, were tested and found to be sufficient for antigen binding. However, features in addition to the rodlike structure may also be necessary for specific binding, because we found that a similar structure present in the RNA of the potato spindle tuber viroid did not allow binding.     

The snRNP 15.5K protein folds its cognate K-turn RNA: a combined theoretical and biochemical study

The human 15.5K protein binds to the 5' stem-loop of U4 snRNA, promotes the assembly of the spliceosomal U4/U6 snRNP, and is required for the recruitment of the 61K protein and the 20/60/90K protein complex to the U4 snRNA. In the crystallographic structure of the 15.5K-U4 snRNA complex, the conformation of the RNA corresponds to the family of kink-turn (K-turn) structural motifs. We simulated the complex and the free RNA, showing how the protein binding and the intrinsic flexibility contribute to the RNA folding process. We found that the RNA is significantly more flexible in the absence of the 15.5K protein. Conformational transitions such as the interconversion between alternative purine stacking schemes, the loss of G-A base pairs, and the opening of the K-turn occur only in the free RNA. Furthermore, the stability of one canonical G-C base pair is influenced both by the binding of the 15.5K protein and the nature of the adjacent structural element in the RNA. We performed chemical RNA modification experiments and observed that the free RNA lacks secondary structure elements, a result in excellent agreement with the simulations. Based on these observations, we propose a protein-assisted RNA folding mechanism in which the RNA intrinsic flexibility functions as a catalyst.     

On the role of rRNA tertiary structure in recognition of ribosomal protein L11 and thiostrepton.

Ribosomal protein L11 and an antibiotic, thiostrepton, bind to the same highly conserved region of large subunit ribosomal RNA and stabilize a set of NH4(+)-dependent tertiary interactions within the domain. In vitro selection from partially randomized pools of RNA sequences has been used to ask what aspects of RNA structure are recognized by the ligands. L11-selected RNAs showed little sequence variation over the entire 70 nucleotide randomized region, while thiostrepton required a slightly smaller 58 nucleotide domain. All the selected mutations preserved or stabilized the known secondary and tertiary structure of the RNA. L11-selected RNAs from a pool mutagenized only around a junction structure yielded a very different consensus sequence, in which the RNA tertiary structure was substantially destabilized and L11 binding was no longer dependent on NH4+. We propose that L11 can bind the RNA in two different 'modes', depending on the presence or absence of the NH4(+)-dependent tertiary structure, while thiostrepton can only recognize the RNA tertiary structure. The different RNA recognition mechanisms for the two ligands may be relevant to their different effects on protein synthesis.     

RNase P: at last, the key finds its lock

Apart from the ribosome, the crystal structure of the bacterial RNase P in complex with a tRNA, reported by Reiter and colleagues recently, constitutes the first example of a multiple turnover RNA enzyme. Except in rare exceptions, RNase P is ubiquitous and, like the ribosome, is older than the initial branch point of the phylogenetic tree. Importantly, the structure shows how the RNA and the protein moieties cooperate to process the pre-tRNA substrates. The catalytic site comprises some critical RNA residues spread over the secondary structure but gathered in a compact volume next to the protein, which helps recognize and orient the substrate. The discussion here outlines some important aspects of that crystal structure, some of which could apply to RNA molecules in general.     

Structure of the Flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing

Yellow fever virus (YFV), a member of the Flavivirus genus, has a plus-sense RNA genome encoding a single polyprotein. Viral protein NS3 includes a protease and a helicase that are essential to virus replication and to RNA capping. The 1.8-A crystal structure of the helicase region of the YFV NS3 protein includes residues 187 to 623. Two familiar helicase domains bind nucleotide in a triphosphate pocket without base recognition, providing a site for nonspecific hydrolysis of nucleoside triphosphates and RNA triphosphate. The third, C-terminal domain has a unique structure and is proposed to function in RNA and protein recognition. The organization of the three domains indicates that cleavage of the viral polyprotein NS3-NS4A junction occurs in trans.