Peter G. Stockley

Aptamers come of age – at last

Nucleic-acid aptamers have the molecular recognition properties of antibodies, and can be isolated robotically for high-throughput applications in diagnostics, research and therapeutics. Unlike antibodies, however, they can be chemically derivatized easily to extend their lifetimes in biological fluids and their bioavailability in animals. The first aptamer-based clinical drugs have recently entered service. Meanwhile, active research programmes have identified a wide range of anti-viral aptamers that could form the basis for future therapeutics.

Structural Insights into the Polymorphism of Amyloid-Like Fibrils Formed by Region 20−29 of Amylin Revealed by Solid-State NMR and X-ray Fiber Diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.

Molecular Responses to Abscisic Acid and Stress Are Conserved between Moss and Cereals.

Promoter elements from the wheat Em gene have been characterized. These elements are inducible by abscisic acid (ABA) and by osmotic stress. In this study, we demonstrated that the same promoter elements function in a distantly related plant species, the moss Physcomitrella patens. Transient and stable expression of the [beta]-glucuronidase reporter gene was used to determine that the heterologous wheat promoter also responds to osmotic stress and ABA in moss. Mutational analysis of the promoter indicated that the mechanism of gene regulation is conserved in both species. Gel retardation and DNase I footprint analyses were conducted to characterize further the interaction of moss transcription factors with the Em promoter. In addition, the synthesis of stress-related polypeptides in moss was observed. The evolutionary significance of these data and the potential for studying the entire ABA perception-response pathway in moss are discussed.

Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid

We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2. The vector has been modified to introduce a unique KpnI restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal beta-hairpin (between residues 15 and 16). Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin. We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties. The chimeric coat proteins self-assemble into largely RNA-free phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro. Such peptide-presenting particles may have a number of biotechnological applications, including use as a cost-effective, synthetic vaccine. We have tested the antigenicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained.

Modus operandi of the bacterial RNA polymerase containing the σ54 promoter‐specificity factor

Bacterial sigma (σ) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative σ factor σ54 confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the σ54 factor makes with the bacterial transcription machinery.

RNA Bacteriophage Capsid-Mediated Drug Delivery and Epitope Presentation

Objective: To use our knowledge of the three-dimensional structure and self-assembly mechanism of RNA bacteriophage capsids to develop novel virus-like particles (VLPs) for drug delivery and epitope presentation. Methods: Site-directed mutagenesis of a recombinant MS2 coat protein expression construct has been used to generate translational fusions encompassing short epitope sequences. These chimeric proteins still self-assemble in vivo into T = 3 shells with the foreign epitope in an accessible location. Covalent conjugation has also been used to generate RNA stem-loops attached to the toxin, ricin A chain, or to nucleotide-based drugs, that are still capable of stimulating self-assembly of the capsid in vitro. These packaged drugs can then be directed to specific cells in culture by further covalent decoration of the capsids with targeting molecules. Results: Chimeric VLPs are strongly immunogenic when carrying either B or T cell epitopes, the latter generating cytokine profiles consistent with memory responses. Immune responses to the underlying phage epitopes appear to be proportional to the area of the phage surface accessible. Phage shells effectively protect nucleic acid-based drugs and, for the toxin construct, make cell-specific delivery systems with LD50 values in culture sub-nanomolar. Conclusion: VLP technology has potential for therapeutic and prophylactic intervention in disease.

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

We have determined the crystal structures, at 2.8 Å 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.

Evidence that viral RNAs have evolved for efficient, two-stage packaging

Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20–30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA–protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein–protein interactions, consistent with the RNA–protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.

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.

Operator interactions by the Bacillus subtilis arginine repressor/activator, AhrC: novel positioning and DNA-mediated assembly of a transcriptional activator at catabolic sites.

We have previously reported the initial characterization of a catabolic operator site (OrocA) for the Bacillus subtilis arginine repressor/activator protein AhrC. Here, we present the characterization by gel retardation and DNase I footprinting of both OrocA and a second catabolic operator site, OrocD. Both operator sites encompass a single recognition site, an ARG box, located immediately upstream of the transcriptional start points, a unique positioning for a transcriptional activator protein. Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around twofold, in their apparent affinities for the protein (Kd′  ≈ 90 nM for OrocA and ≈ 190 nM for OrocD). This difference may result from the lower match to the ARG box consensus of the OrocD site. Both catabolic operators show evidence for co‐operative binding with respect to protein concentration. Determination of the sequences of two AhrC catabolic operator sites, in combination with the three such biosynthetic sites, has allowed the derivation of an improved B. subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t. This is not identical to the Escherichia coli consensus operator for the AhrC homologue, ArgR, which may explain the only partial cross‐functioning of these proteins in vivo. The OrocA site is adjacent to a sharp, stable bend located 5′ to the catabolic operator. Circular permutation analysis has been used to determine the relative angle of bend (≈ 50°), its location and the effect of adding magnesium ions and/or AhrC protein. Protein binding increases the relative bend angle to ≈ 85°. Bending is shown to be associated with a number of A‐tracts in the upstream sequence. However, altering the phasing of the A‐tracts has little effect on the affinity for AhrC. Truncation and competition experiments have been used to investigate the possible role of sequences flanking the operator on affinity. Very surprisingly, the affinity of the OrocA site appears to increase in the presence of excess, specific competitor fragment, i.e. the system shows anti‐competitive effects. Competition is restored at high molar excesses of specific fragment over the protein. We propose a novel model for the assembly of a higher affinity form of AhrC at operator sites that is consistent with both the apparent co‐operativity of binding and the anti‐competitive effects. These data suggest that the molecular interactions occurring between the prokaryotic arginine‐regulatory proteins and their operators may be more complex than is generally appreciated.