A Look At The Picornaviruses Biology Essay

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The picornaviruses are a family of small icosahedral animal viruses that contain single-stranded RNA genomes of positive polarity.1 A significant feature of viral RNA molecules is that they are composed of unusually long 5'-untranslated regions (5'-UTR), and as a consequence comprise of an extensive secondary structure encapsulated by a non-enveloped icosahedral capsid.2 The precise role and mode of recognition of such secondary structures are still unclear at this stage. It is known that the 5'-untranslated region within the genome of a positive-stranded RNA virus, contains prominent, high-order structural elements required for viral RNA replication.3 As a consequence, members of the family Picornaviridae, including both the Foot and Mouth Disease Virus (FMDV), and the encephalomiocarditis virus (EMCV), initiate translation via an Internal Ribosome Entry Site (IRES) element present within the 5'-UTR.2 A study conducted by Jackson & Kaminsk has confirmed that a picornavirus IRES element spans about 450 nt of the 5'-UTR viral RNA.4

1.2 Introduction to IRES Elements

The IRES is composed of a multitude of structured mRNA regions which with the aid of cellular proteins, ensure the facile attachment of an internal AUG codon to a ribosome, as part of the process of protein synthesis.2 In eukaryotic organisms, translation, the final stage of protein synthesis, is initiated only at the 5' end of the mRNA molecule. This feature can be attributed to a 5' cap recognition process; an essential requirement for the formation of an initiation complex required for protein synthesis.5 It is common for the IRES element to allow translation of the RNA virus to occur in a cap-independent manner.6


Prominent characteristics of the FMDV IRES include the presence of a substantially long RNA region comprising of 462 nucleotides which is thought to fold into five distinct domains (named G to L).2 As observed in Figure 1, the FMDV IRES is also composed of a multitude of stable step-loop structures, all of which are phylogenetically conserved.3 The central and largest domain is attributed to be domain I. A series of computational studies conducted by Lopez de Quinto have established the presence of 210 residues within this domain; a feature which ensures that the residues require a stem-loop structure within an apical region.2

Figure . Secondary Structure of FMDV IRES. The C-rich region comprises of the 15-mer sequence being considered for analysis. Also observed is the GNRA motif responsible for the structural integrity of the FMDV IRES.2

The apical region is also the location of an essential, conserved GNRA tetraloop motif; one that is essential for IRES activity. This motif is attributed to contribute to the stability of the structure of RNA, via the generation of stabilising interactions between the tertiary structure and the distant residues of the FMDV IRES. Recent studies conducted by Miragall and Martinéz-Salas, have concluded that the structural organisation of the FMDV IRES is dependent upon the integrity of this GNRA motif.2 In addition to the apical region, the central domain also comprises of a proximal region; a region of the IRES predicted to fold into a stem interrupted by several buldges.3

1.2.2 The Untranslated Regions of FMDV RNA

Recent studies published in the literature, have demonstrated that the untranslated regions situated at either end of positive-stranded viral RNAs within the FMDV picornavirus, have a dominant role in the control of gene expression.7 The 5'- UTR is composed of an S region at the 5' terminus of the FMDV RNA. It is thought to adopt a characteristic cloverleaf structure, but its function is however, unknown. It is understood to be a requisite for RNA replication. The 3'-UTR of FMDV RNA comprises of both a poly(A) tract and two stem-loops. These are essential for both infectivity and replication.4

Serrano and Fernandez have confirmed that the 3' terminus of an FMDV genome demonstrates two succinct strand-specific long-range RNA-RNA interactions, one with the S region and the other with the IRES element.8 This phenomena has made it possible to conduct preliminary free-energy calculations on RNA sequences, to provide support for a stable, well folded structure for predicted motifs, and as a consequence ensure NMR studies are possible.

1.3 NMR Studies of the EMCV IRES

The FMDV IRES is predicted to comprise of a complex secondary structure, similar to that of the EMCV IRES. Within the apical region of the I domain, and within the J and K domains, there are some completely identical regions.2 The 3D structure of the highly conserved and mutationally sensitive RNA secondary structural motif, spanning residues G523-C595 within the IRES element of EMCV virus has already been determined via NMR studies and computational methods.3 In combination, the studies have confirmed the folding of the motif into a "hammerhead" type structure (Figure 2).9 Such successful studies confirm the possible application of NMR studies and computational methods on alternate IRES sequences such as the 15-mer FMDV IRES motif in question.

Figure . The proposed 'hammerhead' RNA Secondary Structural Motif of the EMCV picornavirus.

1.4 Protein Synthesis

Protein synthesis comprises of two major steps: transcription and translation. The initiation of protein synthesis in viral RNA requires the 3' portion of the FMDV 5'-UTR. The discrete features of the FMDV 5'-UTR, however, make it improbable for the FMDV RNA to be translated by a classic cap-dependent translation mechanism.4 Pelletier and Sonenberg were able to demonstrate that the Picornavirus FMDV 5'-UTR was able to direct protein synthesis unusually, via a cap-independent mechanism, after the primary stage of Transcription.3

1.4.1 Transcription

Transcription, the initial stage of protein synthesis, is defined as the synthesis of an RNA molecule from a DNA template. It comprises of three main events:

Initiation: The primary stage entails the binding of the RNA polymerase enzyme onto the duplex of DNA, at a specific sequence ascribed to as the promoter.

Elongation: The second stage involves the covalent addition of nucleotides to the 3' end of the growing polynucleotide chain. This subsequently involves the development of a short stretch of transiently single-stranded DNA for use in the final stage of transcription.

Termination: The final stage entails recognition of the transcription termination sequence. The RNA Polymerase enzyme is also released at this stage.10

1.4.2 Translation: Cap Dependent and Cap Independent Initiation

Eukaryotic translation, the final stage of protein synthesis, is the course by which messenger RNA (mRNA) is translated into proteins in eukaryotic organisms. It comprises of three stages including initiation, elongation and termination.11

Initiation: The majority of eukaryotic mRNA molecules require the cap-binding complex elF4F in order to allow efficient initiation of translation. A ribosomal scanning process from the capped 5' end of the mRNA to the initiation codon, enables the process to occur. As previously stated, initiation can occur either in a cap-dependent or in a cap-independent manner.10

The IRES approach remains the most prominent method of study for the cap-independent mode of translation initiation in eukaryotic organisms.11 Cap-independent translation differs from cap-dependent translation, in the sense that it does not necessitate the ribosome to commence the scanning process from the 5' end of the mRNA cap until the start codon. This enables the ribosome to move to an alternate start site with the aid of an IRES trans-acting factor (ITAF). This effectively makes the requirement to scan from the 5' terminal of the untranslated region of the mRNA, obsolete.10

1.4.3 The Structure and Role of the Ribosome

The ribosome is a large ribonucleoprotein; an essential component of the overall protein synthesising system.11 The reversible dissociation of both the 30S (small) and 50S (large) subunits during protein synthesis enable the attachment of a specific amino acid to a specific transfer RNA (tRNA) molecule. The well defined A site (aminoacyl-tRNA site) and P site (peptidyl-tRNA site) are the two tRNA binding sites on the ribosome. Recent advances in the literature have also established the presence of a third site, called the E site (Exit site), also thought be located on the 50S subunit (Figure 3).10 The ribosome plays a dominant role in the elongation stage of translation.

Figure . Schematic Representation of a ribosome containing both the 30S (small) subunit and 50S (large) subunit. The A site and P site are the two well-characterised tRNA binding sites on the ribosome.12

1.4.4 Translation: Elongation

In this stage, amino acids are added to the growing polypeptide chain as each tRNA delivers its amino acid. The transfer of the amino acid from tRNA to mRNA, entails movement from the P site to an A site. Subsequently, the peptidyl tRNA vacates the A site and moves to the P site. This leaves the A site available for the next amino acid-carrying tRNA.11

1.4.5. The Structure and Role of Transfer RNA (tRNA)

The tRNA molecule plays a dominant role in protein synthesis. The tRNA molecule comprises of a common sequence (....CCA) at the acceptor 3'- termini. During in the concluding stages of protein synthesis, the ribose of the 3'- terminal A residue develops an ester linkage with an amino acid. All tRNA molecules encompass a highly conserved secondary structure (cloverleaf) within which a multitude of base paired stems are separated by a single stranded loop. The anticodon loop pairs with a specific mRNA, and subsequently targets a specific amino acid (Figure 4).10


Figure . Clover Leaf Secondary Structure of tRNA.13

The initial two nucleotides in the tRNA anticodon loop are necessary for the decoding of the mRNA codon into a specific amino acid. However, the final nucleotide in the anticodon is less stringent in its base-pairing to the codon. It is often denoted to as the "wobble" base. Thereby, the degeneracy of the genetic code enables more than one codon to specify a single amino acid. Subsequently the anticodon of a tRNA molecule can pair with more than one mRNA codon and still provide the specific sequence for a single amino acid.10

1.4.6 Translation: Termination

In the final stage of protein synthesis, elongation of the polypeptide chain is terminated when the ribosomal unit encounters a stop codon (UAA or UAG). The newly assembled polypeptide is released from the ribosome though reversible dissociation, releasing both the polypeptide and the mRNA molecule.11

1.5 Nucleic Acids

Nucleic acids are extended, thread-like polymers, composed of a linear array of monomers called nucleotides. They make up the important biological macromolecules essential for life and are present in all living organisms. There are two types of nucleic acids: deoxyribose nucleic acid (DNA), and ribose nucleic acid (RNA); members of a family of biopolymers.14

The experimental studies conducted upon nucleic acids have resulted in the unravelling of substantial developments within both medicine and biological studies. They have formed the basis for developments in the Human Genome, biotechnology, as well as the pharmaceutical industry.15

1.5.1 Nucleosides and Nucleotides

The basic component of a polymeric nucleic acid is the nucleotide. Nucleotides are the phosphate esters of nucleosides, both of which are components of RNA and DNA. More specifically, RNA is made up of ribonucleotides, while in contrast the monomers of DNA are 2'-deoxribonucleotides. Each nucleotide monomer contains a pentose sugar, phosphate residue, and a nitrogenous organic base (Figure 5).16

Figure . A Nucleotide triphosphate unit comprising of a Pentose sugar, Organic base and triphosphate moiety found within RNA (DNA).

In nucleosides, the bases are attached from the ring nitrogen to carbon-1 of a pentose sugar. Within RNA, the pentose is attributed to be D-ribose which is locked into a five-membered furanose ring by the bond from C-1 of the sugar to N-1 of the respective base. This bond is on the equivalent side of the sugar ring as the C-5 hydroxymethyl meoity and is defined as a β - glycosidic linkage.

The carbons to which the phosphate groups are attached are the 3'-end and the 5'-end carbons of the sugar. This is in accordance with conventional nomenclature, and in essence, gives nucleic acids directionality.17

1.5.2 Base Pairing

The major bases are monocyclic pyrimidines and bicyclic purines (Figure 6). The major purines are adenine (A) and guanine (G). They are found in both DNA and RNA. In contrast the major pyrimidines are thymine (T), cytosine (C) and uracil (U).10

Figure . The Monocyclic Purine (adenine and guanine), and bicyclc pyrimidine (thymine, cytosine and uracil) bases.

In canonical Watson-Crick DNA base pairing, adenine forms a base pair with thymine, while cytosine forms a base pair guanine (Figure 7). In RNA, thymine is replaced by uracil.11 There may also be certain instances where an alternate hydrogen bonding pattern gives rise to a complex but functional tertiary structure. Significant examples of this phenomenon include the wobble base pair and Hoogsteen base pair, both of which are prominent in RNA.18

Figure . The Watson Crick Base Pairs: adenine binds to thymine, while guanine binds to cytosine. The red hydrogen atoms indicate the presence of hydrogen bonds.

Purines are only complementary with pyrimidines. Pyrimidine-pyrimidine base pairings are deemed to be energetically unfavourable, as the distances between the bases do not permit hydrogen bonding. Moreover, purine-purine base pairings are also energetically unfavourable as the close proximity of the bases lead to steric repulsion.16

The G-U base pair, often attributed to as a mismatch, is particularly prominent in RNA. It contains two hydrogen bonds. Its high probability is attributed to the existence of the wobble base pair; a phenomenon proposed by Francis Crick to clarify the degeneracy of the genetic code.10

1.6 The Chemical Composition and Physical Properties of DNA

The serendipitous discovery of the right handed double helical model of DNA by Watson and Crick represents a significant breakthrough for modern science (Figure 8). The anti-parallel strands on the double helix encompass the genetic information vital for the development and reproduction of all living organisms.19


Figure . The DNA Double Helix.

The DNA backbone, in essence, comprises of alternating phosphate and sugar groups. The sugar residues are joined to the phosphate groups through phosphodiester bonds.16

1.6.1 The Primary Structure of DNA

Regular DNA comprises of a primary structure in which each nucleoside is attached by a phosphodiester from its 5'-hydroxyl functionality to the 3'-hydroxyl moiety of the adjacent nucleoside, and by a second phosphodiester from its 3'-hydroxyl moiety to the5'-hydroxyl functionality of its alternate neighbor (Figure 9).21

It thereby follows that the unique nature of any DNA primary structure is attributed exclusively to the sequence of its bases.

Figure . The Primary Structure of DNA.

1.6.2 The Secondary Structure of DNA: A- DNA and B-DNA

Recent advances in diffraction studies have enabled the identification of two distinct conformations for the DNA duplex. A multitude of literature sources have identified the highly crystalline A-DNA as the favoured form at low humidity. On the contrary, at high humidity, the dominant structure has been defined as B-DNA (Figure 10).10


A-DNA closely follows the model developed by Watson and Crick, whereby under conditions of minimal water and high salt concentration, the anatomy of the DNA structure displays an anti-parallel, right handed double helix, in which the base pairs are tilted and displaced toward the minor groove.22 X ray diffraction studies conducted by Rich et al. have revealed that the bases are displaced 5Å away from the helix axis. A remarkable feature, however, is the C3'-endo-pucker in the furanose ring and the anti-conformation of the glycoside which develops a 5.4Å P-P separation among adjacent intrastrand phosphates.10


In contrast, B-DNA is most common form of DNA in living organisms; it is a form in which the DNA duplex twists in a right-hand direction.22 The minor and major groves exhibit similar levels of depth in comparison to A-DNA, however, the bases stack above their neighbours in the equivalent strand and perpendicular to the helix axis. B-DNA also encompasses an anti-conformation in the glycoside, but the C2'-endo-pucker in the furanose ring is dominant.10

Figure . The Structure of A-DNA (Left), B-DNA (Middle) and Z-DNA (Right).23

1.6.3 Sugar Pucker

The compact shape of the nucleotides requires details of their conformational structure to be described by torsion angles. These torsion angles, however, are interdependent and thereby an alternate set of parameters are required for the shapes of nucleotides to be described accurately. One such parameter is the sugar pucker, in which the major displacement of the 2' and 3' carbons form the medium plane of C1-O4-C4 are identified. In order to minimize the non-bonding interactions between substituents, the furanose rings are twisted out of plane. Within such a phenomena, if the endo-displacement of C-2' is of a greater magnitude than the exo-displacement of C-3', the conformation is attributed to be C2'-endo as in the case of B-DNA (Figure 11).10

Figure . The C2'-endo and C3'-endo Sugar Puckers.

1.6.4 Major and Minor Groves

The winding of the DNA strands, results in the formulation of gaps among each set of the phosphate backbones. Due to this phenomenon, there are two grooves twisting around the surface of the double helix (Figure 12).24

Figure . The G-C Canonical Watson-Crick base pair. Positions of the minor and major grooves are indicated. The glycosidic sugar-base bond is shown by the red bold line; hydrogen bonding between the two bases is shown in dashed lines.

The major grooves are 22Å wide, and the minor grooves are 12Å wide. The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. The major grove is richer in base substituents, while the minor groove comprises of hydrophobic hydrogen atoms of ribose groups forming its walls.16 Subsequently, proteins akin to transcription factors can bind to specific sequences in double-stranded DNA. Both grooves provide distinctly different environments which are important for recognition and binding.10

1.6.5 The Process of DNA Replication

DNA replication occurs via a semi-conservative mechanism; a phenomenon where each DNA strand composed of a specific nucleotide, is used as the template for the production of a complementary DNA strand (Figure 13).25

The process is initiated by the unwinding of the DNA duplex. This enables the weak hydrogen bonds between the complementary strands within the parent DNA helix to be broken; a process catalysed by the enzyme DNA ligase.10

Figure . The Process of Semi-Conservative Replication.27

The resultant structure is attributed to be known as the replication fork. Within this entity, the strands are separated to an extent in which the bases are exposed. This enables new hydrogen bonds to be formed with the aid of the DNA Polymerase enzyme. Subsequently, RNA primase binds to the initiation point of the parent chain. This feature enables the RNA nucleotides to bind to the DNA nucleotides via the formation of hydrogen bonds between the base pairs.25

The process of replication is different within the two anti-parallel strands of DNA. The leading strand is one in which the nucleotides are continually added to the 3' termini. In contrast, a lagging strand has to be created for the alternate complementary strand, as it exposes a sugar rather than phosphate moiety.10 The lagging strand is thereby one which is synthesized in reverse of the original direction of replication. It also leads to the creation of short sequences between the sites of two RNA primers, known as Okazaki fragments.26 The actions of both DNA polymerase, which adds the complementary nucleotides between the gaps, and DNA ligase, which adds the phosphates to complete the sugar-phosphate backbone enables the final stage of termination to begin.25 Overall, the process creates a double helix comprising of one old and one new DNA strand in a self-complementary sequence.10

1.6.6 The Structure of RNA

In contrast to DNA, RNA occurs typically as a single polynucleotide chain. Nevertheless, by virtue of the inherent ability of RNA to make up different conformations, countless RNA molecules exist in an intricate, defined structure.28

The RNA molecule comprises of a short double-helical region which is coupled by single-stranded stretches. Thereby, the helical hairpin region can form as the anti-parallel orientation of a number of complementary sequences; originate in different parts of the RNA chain. One such example of this phenomenon can be found in the secondary structure of tRNA as previously discussed.10

The preservation of RNA structure can be dependent upon a multitude of factors. These include salt concentration, pH, temperature, and the presence of specific ions (e.g. Mg2+).29

1.6.7 The Role of Mg2+ ions in RNA Stability

The structural integrity and biological activity of RNA is dependent upon the identity concentration of counter ions present within solution. The close packing of phosphate anions on the RNA backbone, result in formation of strong electrostatic repulsive forces and can lead to the unwinding of the RNA polynucleotide strand.30 The presence of divalent cations, such as magnesium, are thereby crucial in reducing the repulsive interactions and in turn are responsible for stabilizing the folded conformation of RNA (Figure 14).31

Figure . Three bound Mg2+ ions in the crystallographic structure of the narrowed major groove of RNA. The lower Mg2+ ions share hydration shells and directly contact anionic phosphate oxygen atoms on the backbone; the central Mg2+ ion forms an outer sphere complex. 32

There are several mechanisms by which magnesium can interact with RNA. These include diffuse binding as well as outer and inner sphere complexes which are principally eminent due to their hydration properties.19 The process of diffuse binding entails fully hydrated divalent ions interacting with nucleic acids, by the use of non-specific long-range electrostatic interactions. These interactions account for the for the "delocalized" counter ion atmosphere, which surround all nucleic acids.32

It has been the development of X-ray crystallography and NMR spectroscopy which has enabled the analysis of macromolecular structure in close detail.33

1.7 Spectroscopic Methods

Spectroscopy is defined as the study of the interaction of electromagnetic radiation with matter. At present, several techniques are being utilised to study the structure of RNA molecules including Molecular modelling and X-Ray Crystallography.

1.7.1 Molecular Modelling

This technique encompasses the multitude of theoretical methods and computational techniques currently being utilised to model and probe the behaviour and underlying stucture of RNA molecules. The benefit of this technique is clearly apparent; computers are able to perform molecular modelling on any reasonably sized system. It enables an atomistic description, allowing several atoms to be considered, during both simulation and calculation.34

1.7.2 X-Ray Crystallography

X-ray Crystallography is an alternate method currently being utilised to probe the underlying structure of RNA. The process entails the scattering of a monochromatic beam of X- ray radiation by the electrons in the atoms of matter, which lie in the beam's path. The wavelength of X-ray radiation (≈10-10 m) is of a dimension analogous to the intermolecular spacing within both biopolymers, and extended crystal structures.35 As a consequence, as the beam interacts with the macromolecule, the interference pattern created, can explicitly determine the location of atoms or ions, with respect to one another. This information can subsequently be extracted by treating the particles as a diffraction grating and applying Bragg's law. The intensities and angles of the diffracted beams enable one to subsequently produce a three-dimensional picture of the density of electrons within the biopolymer in question.36

1.8 Nuclear Magnetic Resonance (NMR)

NMR can be utilsied by chemists to study chemical structure, using simple one-dimensional methods. Information about the dynamics, structure, and interactions with analogous RNA molecules and proteins can be extracted for RNA molecules composed of up to 100 nucleotides.37

The NMR phenomenon entails the interaction of magnetic nuclei with an external magnetic field. The interaction of the magnetic moment with an external magnetic field is termed as the Zeeman interaction (Figure 15).35NMR

Figure . Two stable energy states α and β in an applied magnetic field B0. Transitions occur at the Larmor frequency ν = γB0/2π.35

1.8.1 Spin-Spin Coupling

The spin-spin interactions of adjoining hydrogen atoms take place though a vicinal bonding relationship.38 In such a circumstance, a neighbouring proton which encompasses a +1/2 spin shifts the resonance frequency of the proton to a higher value (up to 7 Hz). Conversely, a proton with a _1/2 neighbouring spin shifts the resonance to a lower frequency. This phenomena is additive if a multitude of neighbouring spins are present. This can be attributed to the notion that the population of two spin states in their entirety are approximately equal; they differ by merely a few parts per million within a strong magnetic field.35

The coupling constant is also known to change with the dihedral angle (φ) between coupled hydrogen atoms. They advocate a relationship, which has been confirmed and clarified by a multitude of established sources within the literature. This relationship is ingeniously articulated by the Karplus eqution (Figure 16).38karplus.jpg

Figure . Graphical Representation of the Karplus Relationship.39

1.8.2 1D NMR


Figure . The Pulse Sequence for 1D NMR.40

1D NMR comprises of two stages: preparation and detection (Figure 17).35 During the preparation stage, the spin system is set within a parameter called the defined state. In contrast, during the detection stage, the signal is recorded. In terms of the pulse sequence the preparation stage involves a 90o pulse which rotates Mz onto the y axis (My).28 Following this pulse sequence each spin precesses according to its own larmor frequency around the z axis and in turn this induces a signal onto the receiver coil. The signal then decays due to the T2 relaxation, and this is known as the free induction decay period. The experiment is repeated several times in order to reduce the signal to noise ratio. The complete set of data is then transferred to the final 1D spectrum.36

1.8.3 2D NMR

In addition to the preparation and detection stages present in 1D NMR, 2D experiments have an indirect evolution time period T1 as well as a mixing sequence (Figure 18). After the preparation stage the spins precess freely for a time T1.37 The mixing stage involves two mechanisms for the transfer of magnetization: scalar or a dipolar interaction. The data is then gathered and during this time the magnetization is labelled as the chemical shift of the second nucleus.30 2D NMR will be of significant importance in analysing the structure of RNA.36

2d nmr

Figure . The Pulse Sequence for 2D NMR.39

Sequence-specific assignments have played a pivotal role in the development of an advanced understanding of both DNA and RNA. A study published by Pullman et al. initiated an increase in interest in the area, and at present there are a multitude of methods present in the literature for obtaining a sequence-specific resonance assignment of a biopolymer in its entirety.37

1.8.4 Nuclear Overhauser Effect Spectroscopy (NOESY)

The Nuclear Overhauser Effect (NOE) arises through the Radio Frequency (RF) saturation of one spin; an effect which causes the perturbation via dipolar interactions with further nucleus spins and one which enhances the intensity of other spins. Dipolar coupling interacts throughout space and as a consequence the steady-state NOE is a very useful instrument to study the conformation of biopolymers.35

For an energy diagram of a two-spin system, both W1S and W1I are attributed to be the quantum transition probability rates for both the observed and saturated spins (Figure 19).

Figure . The Energy and Transitions in a Two-Spin System.

Moreover, both W0 and W2 are the probability rates for zero and double quantum transitions. The W1I and W1S rates are responsible for the spin-lattice relaxation process.41

A difference in population is created across the energy levels when an atom with spin I is saturated with RF. This population difference is different from the one created in the unperturbed state, and thereby manifests the Nuclear Overhauser Effect at unsaturated spin S.35 The pulse sequence for the NOESY experiments to be conducted on the biopolymer sequences is outlined below (Figure 20). NOESY

Figure . The NOESY Experiment Pulse Sequence.42

The structural analysis of intramolecular NOE within both DNA and RNA are of significant importance. NOESY experiments provide signals which correspond to two hydrogen atoms located at a shorter distance than approximately 5.0Å apart in the bioplolymer chain. This technique, in combination with sequence-specific assignments enables specific distance constraints to be attributed to precise sites within both DNA and RNA. NOE experiments are thereby vital as they provide the basis for a systematic procedure towards spatial structure determination within polypeptide chains.35

1.9 Proposal and Hypothesis

To start with, we propose to revise the IRES element of an FMDV picornavirus. We have decided to determine the 3D structure of a highly conserved and sensitive RNA motif (15mer). This secondary structure has been predicted to fold into a stem loop type structure. Herein, we also intend to carry out a 1D and 2D NOESY NMR structural investigation of this motif, in order to confirm its predicted structure. Preliminary free-energy calculations offer support for a stable structure for the predicted motif. At present there is an absence of commercially available drug therapies which can be utilised against the diseases caused by the picornavirus. The results of the study could potentially provide a noteworthy opportunity to design drugs against the FMDV picornavirus.