The blueprint of life is stored at the core of each cell in a nucleic acid. 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, whether animals, plants or viruses. There are two types of nucleic acids, deoxyribose nucleic acid, (DNA) and ribose nucleic acid (RNA); members of a family of biopolymers. All living cells are composed of both DNA and RNA. On the Contrary, viruses only contain DNA or RNA; rather than both.
As a nucleic acid is unbranched, it can comprise of both a linear or circular structure. Significant examples include mitochondrial DNA or Bacterial Chromosomes, which usually embrace a circular double-stranded structure. In contrast, the chromosomes contained within a eukaryotic nucleus are typically linear double-stranded DNA molecules.
The experimental studies of 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.
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The basic component of a polymeric nucleic acid is the nucleotide. Nucleic acids can vary in size, but they are generally very large molecules. DNA molecules are attributed to be the largest molecules known. They range in size from the 21 nucleotides found within small RNA molecules, to the human chromosome which contains 247 million base pairs. Nucleotides have a dominant role in metabolism as they provide the source of chemical energy (ATP or GTP) that is required to partake in cellular signaling. Nucleotides are also integrated within the imperative cofactors of enzymatic reactions, such as coenzyme A and FAD.
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.
The major bases are monocyclic pyrimidines or bicyclic purines. The major purines are adenine (A) and guanine (G) and are found in both DNA and RNA. The major pyrimidines are cytosine (C), thymine (T), and uracil (U).
In nucleosides, the purine and pyrimidine bases are attached from a ring nitrogen to carbon-1 of a pentose sugar. In RNA, the pentose is D-ribose which is locked into a five-membered furanose ring by the bond from C-1 of the sugar to N-1 of Cytosine or Uracil. This bond is on the equivalent side of the sugar ring as the C-5 hydroxymethyl meoity and is defined as a Î² - glycosidic linkage. Nucleosides can in essence be phosphorylated through specific kinases within the sugar's primary alcohol group to produce a nucleotide.
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.
1.2 Base Pairing
Two nucleotides located on adjacent complementary DNA or RNA strands, attached via hydrogen bonds, are termed as a base pair. In the canonical Watson-Crick DNA base pairing, adenine (A) forms a base pair with thymine (T), while cytosine (C) forms a base pair guanine (G). In RNA, thymine is replaced by uracil (U). 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 pre-eminent in RNA. Francis Crick proposed the existence of the 'wobble' base-pair, to clarify the degeneracy of the genetic code. This phenomenon calls for a single base in the 5'-anticodon position of tRNA to be able to recognize either of the pyrimidines bases or, in contrast, either of the purines as its 3'codon base partner.
It is important to note that base pairing is the precise mechanism by which codons on messenger RNA (mRNA) are recognized by the anticodons on transfer RNA during translation.
In essence, the mutual recognition of Adenine by Thymine and of Cytosine by Guanine entails the use of hydrogen bonds to ascertain the fidelity of both DNA transcription and translation processes. The N-H moieties which are situated on the bases are established hydrogen-bond donors. On the contrary the sp2 -hybridized electron pairs located on the oxygen atoms of the base C double bond O groups and on the ring nitrogens are more established hydrogen-bond acceptors than the oxygens of either the pentose or the phosphate.
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Specific base pairing is a key feature of the Watson and Crick model of DNA. Both complementary base pairs are structurally similar. In DNA, the amount of guanine is equal to cytosine and the amount of adenine is equal to thymine. Furthermore, the length of each specific base pair is equivalent. They all fit uniformly among the two phosphate backbones.
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. In contrast purine-purine base pairings are also energetically unfavourable as the close proximity of the bases lead to steric repulsion. Energetically favourable combinations include GT and AC. Although these pairings are mismatches, the specific pattern of hydrogen bonding between the donor and acceptors do correspond. The GU pairing is particularly pre-eminent in RNA; it contains two hydrogen bonds. Its high probability is attributed to the existence of the wobble base pair.
The base stacking interactions in both DNA and RNA are due to short-range exchange repulsion, dispersion, and electrostatic interactions which all contribute to stability. In this instance, the Guanine: Cytosine base pair has a very favourable interaction with adjacent bases due to stacking interactions. These base stacking effects are particularly significant in the secondary structure and tertiary structure of RNA; RNA stem-loop structures are stabilized by base stacking in the loop region
The Chemical Composition and Physical Properties of DNA/RNA
The structural similarity of the Adenine: Thymine and Cytosine: Guanine base pairs, led to the development of the double helix model of DNA as proposed by Watson and Crick. The Hydrogen bonds at the foundation of the double helix provide the methodology to successfully unzip the two complementary strands of DNA; to aid the process of DNA replication.
The DNA backbone strand comprises of alternating phosphate and sugar groups. The sugar that is present in DNA is 2-deoxyribose; which is also known as a pentose consisting of a five carbon structure. Within this structure the sugar residues are joined to the phosphate groups through phosphodiester bonds. Within a double helix the nucleotides in one strand run in an anti-parallel manner. These asymmetric ends of DNA are known as the 5' and 3'ends. The 3' end consist of a terminal hydroxyl group and the 5' prime consist of a terminal phosphate group. The bases are attached to the phosphate groups.
The key difference between DNA and RNA is the sugar 2-deoxyribose within DNA which is replaced by a pentose sugar ribose RNA. The formation of the DNA backbone can be attributed to the twin helical strands. However, an additional double helix may originate within the grooves amid the strands, adjacent to the base pairs. This may provide a binding site.
The double helix is recognized to be in the form of a right-handed spiral. 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. The major grooves are 22 Å wide and the minor grooves are 12 Å 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. 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.
Figure . The canonical Watson-Crick base pair, shown as the G-C pair. Positions of the minor and major grooves are indicated. The glycosidic sugar-base bond is shown by the bold line; hydrogen bonding between the two bases is shown in dashed lines.
The past decade has seen significant improvements in the understanding of the structure known as the hairpin loop. Stem-loop intramolecular base pairing is a pattern which can be found within single-stranded DNA or, more universally, in RNA. The structure is as a result of two regions of an equivalent anti-parallel polynucleotide strands being read in opposite directions, which base-pair, in order to generate a duplex "stem" connected to a single-stranded loop. It is the key building block of a multitude of RNA secondary structures.
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Hairpins not only provide a model system for studying DNA unzipping, but are also the principal motif of secondary structure in RNA. They play an invaluable role in both gene transcription and regulation.
Figure . Hairpin
The multitude of biological functions demonstrated by an RNA molecule can be attributed to the three-dimensional complexity of RNA structures. 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.
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. In this case the 5'-end of the tRNA forms a helix with nucleotides positioned in close proximity to the 3'terminus. As a result the double-helical structure contains not only the standard A:U and G:C base pairs, but also a multitude of energetically less stable G:U base- pairs.
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+).
Role of Mg2+ Ions
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 which can result in the unwinding of the RNA polynucleotide strand. 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.
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.
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" counterion atmosphere, which surround all nucleic acids. The strong electrostatic field neighbouring an RNA molecule can consequently result in the accumulation of diffusely bound ions in pockets of negative electrostatic potential, created by the irregular shape of the molecular surface. However, in some cases, ions become trapped in these electrostatic potential wells; such that their thermal translational energy is not sufficient for them to continue their random motion. These electrostatically bound ions lead to the formation of an "outer sphere" complex. They are thought to retain all but their innermost hydration layer.
Figure . Three bound Mg ions in the crystallographic structure of the narrowed major groove of the loop E fragment of the 5SRNA. The lower Mg ions share hydration shells and directly contact anionic phosphate oxygens on the backbone; the central Mg ion forms an outer sphere complex; and the uppermost ion makes a single direct inner sphere with the backbone.
It has been the development of X-ray crystallography and NMR spectroscopy which has enabled the analysis macromolecular strucutre of in close detail. Structures ranging from tRNA species along with dinucleoside phosphates through oligonucleotides have been probed by these techniques.
1.3 Molecular Modelling
The past decade has seen an increase in the use of molecular modelling to probe the underlying structure of RNA. This technique encompasses the multitude of theoretical methods and computational techniques, which are currently utilised to model the behaviour of RNA molecules. The benefits of this technique are clearly apparent; computers are able to perform molecular modelling of any reasonably sized system. It enables an atomistic description of the molecular system; the complexity of the system is reduced. This allows sseveral additional atoms to be considered during both simulation and calculation.
1.3.1 X-Ray Crystallography
X-ray Crystallography is an alternate methodology to probe the underlying structure of macromolecules including proteins and RNA. The process entails the scattering of a monochromatic beam of X- ray radiation by the electrons in the atoms of matter in the beam's path. This interaction occurs because the wavelength of X-ray radiation (â‰ˆ10-10m) is of a dimension analogous to the intermolecular spacing within both molecules, and extended crystal structures. The interference pattern produced when a beam interacts with the macromolecule can explicitly determine the location of atoms or ions, with respect to one another. The information is subsequently extracted by treating the particles as a diffraction grating and applying Bragg's law. From the angles and intensities of these diffracted beams, one can produce a three-dimensional picture of the density of electrons within the macromolecule.
Spectroscopy is defined as study of the interaction of electromagnetic radiation with matter. Nuclear magnetic resonance spectroscopy entails the use of the NMR phenomenon to study the physical, chemical, and the biological properties of matter. This form of spectroscopy is consistently used by chemists to study chemical structure, using simple one-dimensional techniques. Two-dimensional techniques are used to determine the structure of more complicated molecules. These techniques are replacing X-ray crystallography for the determination of protein structure. This is illustrated by the fact that nearly half of all current RNA structures were determined by using NMR techniques. Information about the structure, dynamics, and interactions with other RNA molecules and proteins can be obtained for RNA molecules up to 100 nucleotides.
The Nuclear Magnetic Resonance 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. In NMR, it is the chemical shift phenomenon which details the dependence of nuclear magnetic energy levels on the electronic environment in a molecule.
1.4.1 Chemical Shift
When an atom is located within a magnetic field, its electrons will circulate in relation to the direction of the applied magnetic field. It is this circulation which results in the small magnetic field at the nucleus which in turn opposes the externally applied field. The electron distribution of the identical type of nucleus (e.g. 1H, 13C, 15N) varies according to local geometry; it entails the consideration of factors such as binding partners, bond lengths, and angles between the bonds.
B = Bo (1-s)
In certain situations, the circulation of the electrons in the aromatic Ï€ orbitals results in a magnetic field at the hydrogen nuclei. This is a phenomenon found in a Benzene molecule and often results in an enhancement of the Bo field; a de-shielding effect.
In theory, nuclei which experience the same chemical environment are termed equivalent. Thereby, nuclei which are close to one another exert an influence on each corresponding magnetic field. When nuclei are non-equivalent; the phenomenon is observable in the NMR spectrum. Furthermore, if the distance between non-equivalent nuclei is less than or equal to three bond lengths, this effect is observable. This effect is called spin-spin coupling.
1D and 2D NMR
1D NMR experiment
1D NMR comprises of two stages: preparation and detection. During the preparation stage, the spin system is set within a parameter called the defined state. During detection 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). 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 once this data is summed up it is then transferred to the final 1D spectrum.
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. After the preparation stage the spins precess freely for a time T1.The magnetization during this stage is labelled as the chemical shift of the very first nucleus. During the period known as the mixing time the magnetization is transferred from the first nucleus to the second. 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.
Process of DNA Replication
DNA replication is the basis for biological inheritance. It is the fundamental process which occurs in all living organisms. The DNA replication sequence starts with the unzipping of the parent DNA molecule, via the breakdown of the hydrogen bonds between the base pairs. Once these bases are exposed they act as template strands in which complementary base pairing can occur on the strand being synthesized. These new stands are assembled from deoxynucleoside triphosphates. The incoming nucleotide is then covalently linked to the free 3' carbon atom on the pentose. During this stage the second and third phosphate is then removed as a molecule of pyrophosphate. The nucleotides are assembled such that they complement the ordered base pairing on the template strand. Therefore each Cytosine on the template guides the insertion of Guanine, and each Guanine guides the insertion of a Cytosine. Once the process is complete two DNA molecules have been synthesized identical to one another and also to the parent molecule. This process is semi-conservative, in that each strand of the original double-stranded DNA molecule, acts as the template for the reproduction of the complementary strand.
Transcription can be defined as the synthesis of an RNA molecule from a DNA template. It has three main events:
The initiation stage entails the binding of RNA polymerase enzyme onto the double-stranded DNA; this step involves a transition to single-strandedness in the region of binding; RNA polymerase binds at a sequence of DNA called the promoter.
The Elongation stage involes the covalent addition of nucleotides to the 3' end of the growing polynucleotide chain; this involves the development of a short stretch of DNA that is transiently single-stranded
The final termination step entaisl recognition of the transcription termination sequence and the release of RNA polymerase
Eukaryotic translation is the course by which messenger RNA is translated into proteins in eukaryotic organisms. It comprises of three stages including initiation, elongation and termination
To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. Initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA.
In bacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. Ribosome shunting, or the ribosomal shunt initiation pathway is an alternate viral mechanism of translation initiation in which ribosomes bind to the mRNA in a normal cap-dependent mode, then jump upstream (5') of the initiator AUG codon.
The Initiation of the translation stage usually entails the interaction of certain key proteins with a unique tag attached to the 5'-end of an mRNA molecule. They are ascribed to as the 5' cap. The protein factors bind to the 40S small ribosomal subunit. The initiation factors created subsequently hold the mRNA molecule in place.
The Cap-Independent Initiation
The IRES approach remains the most prominent method of study for the cap-independent mode of translation initiation in eukaryotes. Cap-independent translation differs from cap-dependent translation as it does not necessitate the ribosome to commence the scanning process from the 5' end of the mRNA cap until the start codon.
2. Elongation - amino acids are added to the growing polypeptide chain as each tRNA delivers its amino acid, forming a complex with elongation factor (EF) and GTP. The amino acid is transferred from the tRNA to the mRNA, moving from the P site to the A site. Next, the peptidyl tRNA vacates the A site and moves to the P site, leaving the A site available for the next amino acid-carrying tRNA. Amino acids are joined by peptide bonds as carboxyl group are added to the 3' OH by an ester bond. The ribosome acts as an enzyme (ribozyme) in the formation of the peptide bond.
3. Termination - elongation of the polypeptide chain ceases when the ribosomal machine encounters a nonsense (stop) codon (UAA, UGA, or UAG). The newly assembled polypeptide is released from the ribosomal machine when the ribosome breaks into its large and small subunits, releasing both the polypeptide and its mRNA.
The picornaviruses are a family of small icosahedral animal viruses that contain single-stranded RNA genomes (~ 8K bases). A salient feature of these viral RNAs is that they are endowed with unusually long 5'-untranslated regions (5'-UTR) containing a high degree of secondary structure. As previously discussed, the precise role and mode of recognition of such secondary structures are unclear at this stage. As a consequence of this feature, initiation of protein synthesis has been shown to take place by a novel mechanism. According to this mechanism, translation of picornaviral RNAs is directed by RNA elements (~ 450 nucleotides) known as internal ribosomal entry sites (IRES) that occur within the 5'-UTR regions. An internal ribosome entry site is a nucleotide sequence that allows for translation initiation in the middle of mRNA sequence as part of the process of protein synthesis. In eukaryotes, translation can be initiated only at the 5' end of the mRNA molecule, since 5' cap recognition is an essential requirement for the formation of the initiation complex.
It has been hypothesized that IRES elements have a distinct secondary or even tertiary structure, but similar structural features at the levels of either primary or secondary structure that are common to all IRES segments have not been reported to date. It is common that IRESes are located in the 5'UTR of RNA viruses and allow translation of the RNAs in a cap-independent manner.
Features of the 5'-UTR:
The 5'-UTR of FMDV RNA comprises of numerous discrete regions. The S-fragment is predicted to fold into a large hairpin structure. Its function is however, unknown. It is understood to be a requisite for RNA replication. The 5'-end of the PV RNA exhibits a characterised cloverleaf structure, which has been known to interact with both viral and cellular proteins. In this case this region is known to be involved in the course of RNA replication. It also exhibits a significant effect on the stability of viral RNA.
Furthermore, the existence of a poly(C) tract within the 5'-UTR is an established feature of the FMDV Virus. In recent times, Mason et al. have confirmed the presence of a stable stem-loop element in the FMDV 5'-UTR. Each of the picornavirus structures contains a conserved motif of AAACA situated within the loop region.
The initiation of protein synthesis process in viral RNA requires the 3' portion of the FMDV 5'-UTR. The discrete features of the FMDV 5'-UTR, however, make it improbable that the FMDV RNA be translated by a classic cap dependent translation mechanism. Despite this consideration, the FMDV RNA is still an efficient template for translation. Features whach are pre-eminent in the vast majority of picornavirus RNA 5'-UTRs include the absence of the cap structure, and the existence of a secondary structure which consist of a multitude of unused AUG codons. Pelletier and Sonenberg (1988) were able to understand the mechanism of picornavirus translation initiation. They were able to demonstrate that the PV 5'-UTR was able to direct cap-independent internal initiation of protein synthesis.
Analogous results were also obtained with the 5'-UTR from EMCV by Jang et al. (1988). The element required for this activity is now usually referred to as an internal ribosome entry site (IRES). Shortly afterwards, it was demonstrated that an element (located immediately upstream of the polyprotein coding region) of about 450 nt within the 5'-UTR of FMDV RNA functioned as an IRES. Similar results were also obtained by Jang et al for the translation of 5'-UTR from EMCV. The element that is responsible for the process to occur via a cap independent mechanism is now known as the IRES (internal ribosome entry site).
The FMDV IRES is predicted to have a complex secondary structure which is very similar to that of the EMCV IRES. The sequence identity between the FMDV and EMCV IRES elements is about 50%, but there are some completely identical regions, particularly within the apical region of the I domain and within the J and K domains. It is assumed that these highly conserved regions will generally reflect critical regions of the IRES. Within the I domain there is a conserved GNRA tetraloop motif which is important for activity. Tetraloop sequences that fit the GNRA consensus are over-represented, on a statistical basis, within structured RNA elements, and it is believed that they play an important role in RNA-RNA interactions and in RNA-protein interactions. Modification of just the 30 A residue within this motif greatly diminishes the activity of either the EMCV or FMDV IRES. Direct RNA-RNA interactions between the I domain of the FMDV IRES and other regions of the IRES have been demonstrated, and evidence suggests that the structural organization of the IRES is dependent on the GNRA motif.
1.4 Proposal and Hypothesis
To start with, we propose to study the IRES element of FMDV virus and have chosen to determine the 3D structure of the highly conserved and mutationally sensitive RNA secondary structural motif (15mer), which has been predicted to fold into a stem loop type structure. Herein, we also intend to carry out a NMR structural investigation of this motif and conduct preliminary free-energy calculations to offer support for a stable, well folded structure for the predicted motif. At present there is an absence of commercially available drug therapies which can be utilsed against the diseases caused by picornaviruses. The results of the study will thereby provide a noteworthy opportunity to design drugs against the FMDV virus RNA.