Structural basis of pyrimidine specificity in the MS2 RNA hairpin-coat-protein complex.

We have determined the X-ray structures of six MS2 RNA hairpin-coat-protein complexes having five different substitutions at the hairpin loop base -5. This is a uracil in the wild-type hairpin and contacts the coat protein both by stacking on to a tyrosine side chain and by hydrogen bonding to an asparagine side chain. The RNA consensus sequence derived from coat protein binding studies with natural sequence variants suggested that the -5 base needs to be a pyrimidine for strong binding. The five -5 substituents used in this study were 5-bromouracil, pyrimidin-2-one, 2-thiouracil, adenine, and guanine. The structure of the 5-bromouracil complex was determined to 2.2 A resolution, which is the highest to date for any MS2 RNA-protein complex. All the complexes presented here show very similar conformations, despite variation in affinity in solution. The results suggest that the stacking of the -5 base on to the tyrosine side chain is the most important driving force for complex formation. A number of hydrogen bonds that are present in the wild-type complex are not crucial for binding, as they are missing in one or more of the complexes. The results also reveal the flexibility of this RNA-protein interface, with respect to functional group variation, and may be generally applicable to other RNA-protein complexes.

Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: identification of a protein conformer unable to bind RNA.

We have investigated the kinetics of complex formation between bacteriophage MS2 coat protein subunits and synthetic RNA fragments encompassing the natural translational operator site, or the consensus sequences of three distinct RNA aptamer families, which are known to bind to the same site on the protein. Reactions were assayed using stopped-flow fluorescence spectroscopy and either the intrinsic tryptophan fluorescence of the protein or the signals from RNA fragments site-specifically substituted with the fluorescent adenosine analogue 2'-deoxy, 2-aminopurine. The kinetics observed were independent of the fluorophore being monitored or its position within the complex, indicating that the data report global events occurring during complex formation. Competition assays show that the complex being formed consists of a single coat protein dimer and one RNA molecule. The binding reaction is at least biphasic. The faster phase, constituting 80-85 % of the amplitude, is a largely diffusion driven RNA-protein interaction (k1 approximately 2x10(9) M(-1) s(-1)). The salt dependence of the forward reaction and the similarities of the on-rates of lower-affinity RNA fragments are consistent with a diffusion-controlled step dominated by electrostatic steering. The slower phase is independent of reactant concentration, and appears to correspond to isomerisation of the coat protein subunit(s) prior to RNA binding (k(iso) approximately 0.23 s(-1)). Measurements with a coat protein mutant (Pro78Asn) show that this phase is not due to cis-trans isomerisation at this residue. The conformational changes in the protein ligand during formation of an RNA-protein complex might play a role in the triggering of capsid self-assembly and a model for this is discussed.

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 A 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.

RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour.

We have probed the effects of altering buffer conditions on the behaviour of two aptamer RNAs for the bacterio-phage MS2 coat protein using site-specific substitution of 2'-deoxy-2-aminopurine nucleotides at key adenosine positions. These have been compared to the wild-type operator stem-loop oligonucleotide, which is the natural target for the coat protein. The fluorescence emission spectra show a position and oligonucleotide sequence dependence which appears to reflect local conformational changes. These are largely similar between the differing oligonucleotides and deviations can be explained by the individual features of each sequence. Recognition by coat protein is enhanced, unaffected or decreased depending on the site of substitution, consistent with the known protein-RNA contacts seen in crystal structures of the complexes. These data suggest that the detailed conformational dynamics of aptamers and wild-type RNA ligands for the same protein target are remarkably similar.

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.

Crystal structures of a series of RNA aptamers complexed to the same protein target.

We have determined the crystal structures, at 2.8 A resolution, of two different RNA aptamers, each bound to MS2 coat protein. One of the aptamers contains a non-Watson-Crick base pair, while the other is missing one of the unpaired adenines that make sequence-specific contacts in the wild-type complex. Despite these differences, the RNA aptamers bind in the same location on the protein as the wild-type translational operator. Comparison of these new structures with other MS2-RNA complexes allows us to refine further the definition of the minimal recognition elements and suggests a possible application of the MS2 system for routine structure determination of small nucleic acid motifs.

Dissecting the key recognition features of the MS2 bacteriophage translational repression complex.

The MS2 RNA operator capsid offers an unparalleled opportunity to study sequence-specific protein-protein and RNA-protein interactions in molecular detail. RNA molecules encompassing the minimal translational operator recognition elements can be soaked into crystals of RNA-free coat protein shells, allowing the RNA to access the interior of the capsids and make contact with the operator binding sites. Correct interpretation of these structural studies depends critically on functional analysis in solution to confirm that the interactions seen in the crystal are not an artefact of the unusual approach used to generate the RNA-protein complexes. Here we present a series of in vivo and in vitro functional assays, using coat proteins carrying single amino acid substitutions at residues which either interact with the operator RNA or are involved in stabilizing the conformation of the FG loop, the site of the major quasi-equivalent conformational change. Variant operator RNAs have been assayed for coat protein affinity in vitro. The results reveal the robustness of the operator-coat protein interaction and the requirement for both halves of a protein dimer to contact RNA in order to achieve tight binding. They also suggest that there may be a direct link between the conformation of the FG loop and RNA binding.

Crystal structures of MS2 coat protein mutants in complex with wild-type RNA operator fragments.

In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.

Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution.

The crystal structure, at 2.8 A resolution, of an RNA aptamer bound to bacteriophage MS2 coat protein has been determined. It provides an opportunity to compare the interactions of MS2 coat protein and wild type operator with those of an aptamer, whose secondary structure differs from the wild type RNA in having a three-base loop (compared to a tetraloop) and an additional base pair between this loop and the sequence-specific recognition element in the stem. The RNA binds in the same location on the coat protein as the wild type operator and maintains many of the same RNA-protein interactions. In order to achieve this, the RNA stem loop undergoes a concerted rearrangement of the 3' side while leaving the 5' side and the loop interactions largely unchanged, illustrating the ability of RNA to present similar molecular recognition surfaces from distinct primary and secondary structures.

The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions.

Crystal structures of two complexes between recombinant MS2 capsids and RNA operator fragments have been determined at 2.7 A resolution. The coat protein of the RNA bacteriophage MS2 is bifunctional; it forms the icosahedral virus shell to protect the viral nucleic acid and it acts as a translational repressor by binding with high specificity to a unique site on the RNA, a single stem-loop structure, containing the initiation codon of the gene for the viral replicase. In order to determine the structure of these protein-RNA complexes, we have used chemically synthesized variants of the stem-loop fragment and soaked them into crystals of recombinant capsids. The RNA stem-loop, as bound to the protein, forms a crescent-like structure and interacts with the surface of the beta-sheet of a coat protein dimer. It makes protein contacts with seven phosphate groups on the 5' side of the stem-loop, with a pyrimidine base at position -5, which stacks onto a tyrosine, and with two exposed adenine bases, one in the loop and one at a bulge in the stem. Replacement of the wild-type uridine with a cytosine at position -5 increases the affinity of the RNA to the dimer significantly. The complex with RNA stem-loop having cytosine at this position differs from that of the wild-type complex mainly by having one extra intramolecular RNA interaction and one extra water-mediated hydrogen bond.

Analysis of phage MS2 coat protein mutants expressed from a reconstituted phagemid reveals that proline 78 is essential for viral infectivity.

A full-length cDNA copy of the RNA genome of bacteriophage MS2 was assembled by the in-frame ligation of the central portion of the genome into a plasmid containing the 5' and 3' ends. Upon transformation of the ligation reaction into Escherichia coli, infectious phage particles were released into the medium. The plaquing ability of the phage produced from the cDNA construct was assessed against various bacterial strains confirming that the bacteriophage produced were male-specific. Sensitivity to RNase in agar overlay was used to confirm that the phage contained RNA. In addition, the phage were unable to infect piliated cells overexpressing MS2 coat protein, a resistance conferred by the binding of recombinant coat protein to the infecting strand of RNA at the replicase initiation region, thus preventing translation of the replicase gene. The phage capsids were visualised after negative staining by transmission electron microscopy, and appeared as spherical particles of approximately 25 nm diameter. The capsid proteins were examined by Western blotting, confirming the presence of a single protein of approximately 14 kDa, which bound anti-MS2 coat protein antibodies. The genomic RNA from single plaques was analysed by reverse transcription-PCR and the presence of the MS2 coat protein gene confirmed by DNA sequencing. The production of replicative MS2 phage from cDNA fragments was used to assess the viability of MS2 coat protein mutants, which had previously been shown to assemble into T = 3 capsid-like particles when expressed in vivo from a bacterial vector. The E76D mutation did not appear to affect phage viability, whilst replacement of the completely conserved P78 residue with asparagine abolished the production of infectious particles, suggesting that P78 may be involved in interactions with the phage maturation protein.

Crystal structures of MS2 capsids with mutations in the subunit FG loop.

The loop between the F and G beta strands (FG loop) of the bacteriophage MS2 coat protein subunit forms inter-subunit contacts around the 5-fold and 3-fold (quasi 6-fold) axes of the T=3 protein shell. In capsids, the loop is found in two very different conformations, one in B subunits, which form the 5-fold contact, and one in A and C subunits, which form the quasi 6-fold contact. One proline residue, Pro78, is strictly conserved in the coat protein of all related bacteriophages, and in the case of MS2 this proline residue is preceded by a cis peptide bond in the B subunit. In order to probe the role of the FG loop in capsid assembly, we have determined the crystal structures of two MS2 capsids, formed by coat proteins with mutations at two positions in the FG loop, P78N or E76D. These mutants show conformational changes in the FG loops that explain the reduced temperature stability of the capsids. The P78N mutant has a normal trans peptide bond at position 78.

Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein.

We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions.

Molecular mechanism of RNA phage morphogenesis.

Recent progress on the molecular mechanism of RNA phage morphogenesis is described. Functional studies, both in vivo and in vitro, are correlated with the latest structural studies on phages, their capsids and the assembly initiation RNA stem-loop.

Crystal structure of an RNA bacteriophage coat protein-operator complex.

The RNA bacteriophage MS2 is a convenient model system for the study of protein-RNA interactions. The MS2 coat protein achieves control of two distinct processes--sequence-specific RNA encapsidation and repression of replicase translation--by binding to an RNA stem-loop structure of 19 nucleotides containing the initiation codon of the replicase gene. The binding of a coat protein dimer to this hairpin shuts off synthesis of the viral replicase, switching the viral replication cycle to virion assembly rather than continued replication. The operator fragment alone can trigger self-assembly of the phage capsid at low protein concentrations and a complex of about 90 RNA operator fragments per protein capsid has been described. We report here the crystal structure at 3.0 A resolution of a complex between recombinant MS2 capsids and the 19-nucleotide RNA fragment. It is the first example of a structure at this resolution for a sequence-specific protein-RNA complex apart from the transfer RNA synthetase complexes. The structure shows sequence-specific interactions between conserved residues on the protein and RNA bases essential for binding.

Effects of amino acid substitution on the thermal stability of MS2 capsids lacking genomic RNA.

The thermal stability of capsids of the bacteriophage MS2, lacking genomic RNA, has been investigated using electron microscopy. Coat protein mutants with amino acid substitutions at residues involved in making contracts at both inter-molecular interfaces and within the coat protein submit are also capable of forming 'empty' capsids of the same size and symmetry as the wild-type protein. Mutations have been characterised which are neutral, deleterious or advantageous in terms of thermal stability. In some cases, the results can be rationalised by reference to the recently refined X-ray crystal structure of the wild-type particle.

Extracellular processing of enamel matrix proteins and the control of crystal growth.

The origin, expression and role of matrix modifying enzymes in dental enamel was investigated by zymography and growth of enamel crystals in vitro. Gelatinase activity of the neutral metalloprotease type was detected at similar molecular weights in enamel organ, enamel and dentine. The activity was present throughout all developmental stages in enamel organ but was dramatically reduced in the maturation stage of the enamel. Activity of the serine protease type directed against enamel matrix was also detected in enamel, particularly in the maturation stage. No such activity was detected in the enamel organ. Phosphatase activity at alkaline pH was demonstrated at similar molecular weights in both enamel and enamel organ. This activity was maximal in the maturation stage. Further experiments showed that both serine proteases and alkaline phosphatase were able to facilitate enamel crystal growth in vitro. Matrix modification via temporally and spatially restricted enzymes may be directly involved in the control of enamel crystal growth and hence in the determination of final tissue architecture.

Control of crystal growth during enamel maturation.

Attempts to promote crystal growth in maturation stage enamel from rat incisors were carried out by incubation in saturated solutions of calcium phosphate. Resulting crystallites were visualised in the TEM and the dimensions of their profiles measured. No crystal growth was observed unless the maturation stage enamel was first pretreated with either 8M urea or sodium hypochlorite to remove residual protein matrix. The results suggest that the protein matrix plays an important role in the control of crystal growth in vivo.