Primary Structure of Nucleic Acids: Chemical and Physical Properties
In this section, we will explore the primary structure of nucleic acids, focusing on their fundamental chemical and physical properties. Understanding these characteristics is crucial as they directly dictate the biochemical and biological functions of and within living systems. We will examine how these properties are intrinsically linked to their roles in information storage, transfer, and utilization. For each subsequent chapter, detailed topics and references to relevant textbook chapters will be provided to facilitate effective and in-depth study.
Chemical Components of Nucleic Acids
Nucleic acids are high molecular weight biopolymers. Complete hydrolysis of a nucleic acid yields three fundamental components:
Nitrogenous Bases: Heterocyclic aromatic compounds classified as either purines (Adenine (A), Guanine (G)) or pyrimidines (Cytosine (C), Thymine (T), Uracil (U)).
Pentose Sugar: A five-carbon sugar, which is either deoxyribose in or ribose in . Deoxyribose differs from ribose by the absence of a hydroxyl group at the 2’ position.
Phosphate Groups: Derived from phosphoric acid. Phosphate groups link the sugar moieties in the nucleic acid backbone and are responsible for the overall negative charge of nucleic acids at physiological pH.
These basic units assemble to form more complex structures known as nucleosides and nucleotides.
Nucleosides and Nucleotides: Building Blocks of Nucleic Acids
Nucleoside Formation: A nucleoside is formed by a condensation reaction between a nitrogenous base and the carbon 1’ of a pentose sugar. This linkage is a N-glycosidic bond, specifically involving the N9 of purines or N1 of pyrimidines.
Nucleotide Formation: A nucleotide is a nucleoside with one or more phosphate groups attached. A nucleotide is formed when a phosphate group is esterified to the sugar component of a nucleoside. This esterification typically occurs at the 5’ or 3’ carbon of the sugar. In the case of ribose, phosphorylation can also occur at the 2’ position.
Phosphorylation of a nucleoside at the sugar moiety transforms it into a nucleotide, the monomeric unit of nucleic acids.
Physicochemical Properties and Biological Significance
The biological functions of nucleic acids are intrinsically linked to their physicochemical properties, which are determined by the molecular characteristics of their nucleotide components. Key properties include:
Delocalized \(\pi\) Electrons and Base Stacking
Nitrogenous bases are aromatic heterocyclic rings containing delocalized \(\pi\) electrons. These electrons are crucial for:
Base Stacking Interactions: The \(\pi\) electrons facilitate stacking interactions between adjacent bases along the nucleic acid chain. These interactions, although individually weak, collectively contribute significantly to the stability of the nucleic acid structure.
Susceptibility to Chemical Modifications: The delocalized \(\pi\) electrons, being relatively reactive, render the bases susceptible to chemical modifications, such as oxidation and alkylation.
Hydrogen Bond Donors and Acceptors
Nitrogenous bases possess functional groups that act as hydrogen bond donors and hydrogen bond acceptors.
Hydrogen Bond Donors: Examples include amino groups (-NH\(_2\)) present on bases like Adenine and Cytosine.
Hydrogen Bond Acceptors: Examples include keto groups (C=O) and nitrogen atoms (=N-) present in bases like Guanine, Thymine, Cytosine, and Adenine.
These groups are critical for:
Complementary Base Pairing: The formation of specific hydrogen bonds between complementary bases (A with T/U, and G with C) is fundamental to the double helix structure of and secondary structures of .
Protein-Nucleic Acid Interactions: Hydrogen bond donors and acceptors on the bases are recognized by proteins that interact with and , such as transcription factors and DNA repair enzymes, enabling sequence-specific recognition.
Role of the 2’-Hydroxyl Group in RNA
The presence of a 2’-hydroxyl group in ribose, which is absent in deoxyribose, imparts distinct properties to compared to :
Structural Differences: The 2’-OH group sterically hinders from adopting a B-form helix, which is typical for . predominantly exists as single-stranded structures or adopts different helical conformations (e.g., A-form helix in double-stranded regions).
Chemical Instability: The 2’-OH group makes more susceptible to hydrolysis, particularly under alkaline conditions (pH \(>\) 8). At elevated pH, the 2’-OH group can participate in an intramolecular nucleophilic attack on the adjacent phosphodiester bond, leading to chain cleavage. This mechanism of degradation is in contrast to , which is stable at pH 8 due to the absence of the 2’-OH group.
The 2’-hydroxyl group in renders it chemically less stable than , especially under alkaline conditions. This is a key distinction between and , influencing their biological roles and longevity within the cell.
Phosphate Group and Negative Charge
The phosphate group, with a pKa around 1, is ionized and carries a negative charge at physiological pH (approximately 7.4). This negative charge is crucial because:
Overall Negative Charge of Nucleic Acids: It imparts an overall negative charge to and molecules.
Electrostatic Interactions with Proteins: The negative charge facilitates electrostatic interactions with positively charged regions of proteins, particularly basic proteins like histones. This electrostatic interaction is often the initial step in protein-nucleic acid binding.
Non-Informational Interactions: While essential for initial binding, the phosphate group’s interaction is not sequence-specific. Specific recognition of nucleic acid sequences by proteins relies on interactions with the hydrogen bond donors and acceptors of the bases.
Chemical Properties and Nomenclature of Nucleobases
Classification Based on Functional Groups: Oxo and Amino Bases
Nitrogenous bases are further classified based on the presence of specific functional groups:
Oxo Bases (Keto Bases): These bases contain keto groups (C=O). Examples include Guanine, Thymine, and Uracil. They are also referred to as ketonic bases or oxo bases.
Amino Bases: These bases contain amino groups (-NH\(_2\)). Examples include Adenine and Cytosine. They are also referred to as aminic bases or amino bases.
For example, Cytosine can be systematically named as 2-oxo-4-amino-pyrimidine, indicating the positions of the oxo and amino substituents on the pyrimidine ring.
Systematic Numbering and Nomenclature
The atoms within the rings of nucleobases are systematically numbered according to IUPAC conventions:
Pyrimidine Bases: Atoms in the pyrimidine ring are numbered from 1 to 6. Numbering starts with the nitrogen atom that is part of the N-glycosidic bond and proceeds clockwise.
Purine Bases: Atoms in the purine ring system are numbered from 1 to 9. The pyrimidine portion of the purine ring is numbered clockwise, and the imidazole portion is numbered counter-clockwise. The N9 atom of purines is involved in the N-glycosidic bond.
Understanding this systematic nomenclature is essential for accurately describing chemical modifications and interactions involving specific atoms of the nucleobases.
Molecular Weights of Nucleotide Components
The molecular weight of a nucleotide varies between 300 and 350 Daltons (Da), depending on the type of pentose sugar and nitrogenous base. Approximate molecular weights of key components are:
Ribose: 212 Da
Deoxyribose: 196 Da
Phosphate Group (PO\(_4^{3-}\)): Approximately 80 Da (contributes consistently to both ribose and deoxyribose nucleotides)
Nitrogenous Bases:
Adenine (A) and Cytosine (C): Lower molecular weights compared to Guanine and Thymine.
Guanine (G) and Thymine (T): Higher molecular weights compared to Adenine and Cytosine.
The slight differences in base molecular weights have biological implications. For instance, in mitochondrial , the two strands are referred to as ‘heavy’ and ‘light’ strands based on their buoyant density in cesium chloride gradients, which correlates with base composition: the ‘heavy’ strand is enriched in guanine and thymine, while the ‘light’ strand is enriched in adenine and cytosine.
Flexibility and Rotational Freedom of Nucleic Acid Chains
Nucleic acid chains exhibit significant flexibility due to the rotational freedom around single covalent bonds within their structure. This flexibility is crucial for their biological functions. Key aspects of flexibility include:
Covalent Bonds and Rotation: Nucleic acids contain several single covalent bonds that allow rotation:
Phosphoester and Phosphodiester Bonds: These bonds in the sugar-phosphate backbone, while strong, permit rotation. Note that the term phosphoester bond refers to the linkage between a phosphate group and a single sugar in a mononucleotide, whereas phosphodiester bond describes the linkage between phosphate and two sugar residues in a polynucleotide chain.
N-Glycosidic Bond: The bond linking the base to the sugar also allows rotation.
Rotational Freedom of Bases: Nitrogenous bases can rotate approximately 360° around the N-glycosidic bond.
Conformational Flexibility of Sugar: The sugar ring itself is not planar and can adopt different conformations, further contributing to flexibility.
Biological Significance of Flexibility: This inherent flexibility is essential for various biological processes, allowing nucleic acids to adopt different conformations required for replication, transcription, folding, and interactions with proteins.
Flexibility of Nucleic Acids:
The flexibility of and molecules, arising from the rotational freedom of their covalent bonds, is crucial for their dynamic interactions and diverse roles in cellular processes. This flexibility enables conformational changes necessary for replication, transcription, and protein binding.
Chemical Instability and Spontaneous DNA Modifications
Inherent Chemical Instability of DNA
, despite being the carrier of genetic information, is inherently chemically unstable. This instability arises from:
Reactive Chemical Groups: The presence of chemically reactive groups and unsaturated bonds within the bases and sugar-phosphate backbone.
Spontaneous Chemical Reactions: is constantly subjected to spontaneous chemical reactions, even under physiological conditions, due to its interaction with water and other cellular molecules.
Major Types of Spontaneous DNA Damage
Spontaneous damage occurs through various mechanisms:
Hydrolysis:
Depurination: Hydrolysis of the N-glycosidic bond between a purine base (Adenine or Guanine) and deoxyribose, resulting in the loss of the purine base and creating an apurinic site (AP site). Depurination is a frequent spontaneous event.
Depyrimidination: Less frequent hydrolysis of the N-glycosidic bond of pyrimidines, leading to apyrimidinic sites.
Oxidation: Reactive oxygen species (ROS) can attack , leading to:
Base Oxidation: Oxidation of nitrogenous bases, for example, guanine oxidation to 8-oxo-guanine (8-oxoG), a mutagenic lesion.
Sugar Oxidation: Oxidation of the deoxyribose sugar.
DNA Strand Breaks: Oxidative damage can also cause breaks in the backbone.
Alkylation: Spontaneous or induced alkylation reactions can add alkyl groups to bases, modifying their structure and base-pairing properties.
Reactive Oxygen Species (ROS) as Primary Damaging Agents
Reactive Oxygen Species (ROS) are significant contributors to spontaneous damage. Major ROS include:
Superoxide Anion (\(\text{O}_2^{\cdot-}\)).
Hydrogen Peroxide (\(\text{H}_2\text{O}_2\)).
Hydroxyl Radical (\(\cdot\text{OH}\)). The hydroxyl radical is particularly reactive and damaging to .
ROS are generated endogenously as byproducts of normal cellular metabolism, especially during oxidative phosphorylation in mitochondria. They can also be induced by exogenous factors like radiation and certain chemicals.
Cellular DNA Repair Mechanisms and Genome Stability
To counteract the constant threat of damage, cells have evolved sophisticated DNA repair mechanisms. These pathways are essential for:
Maintaining Genome Integrity: Repair systems continuously scan the genome and repair lesions, thus maintaining the integrity of genetic information.
Preventing Mutations: Efficient repair reduces the frequency of mutations that could arise from unrepaired damage.
Frequency of DNA Lesions vs. Mutation Rate
There is a significant difference between the frequency of lesions and the spontaneous mutation rate:
High Lesion Frequency: It is estimated that a typical human cell experiences thousands of spontaneous lesions per day (e.g., approximately 10,000). Depurination alone accounts for a significant fraction of these lesions.
Low Mutation Rate: Despite the high lesion frequency, the spontaneous mutation rate is remarkably low, approximately 2-3 mutations per cell cycle.
Efficiency of Repair Systems: This discrepancy highlights the remarkable efficiency of repair systems in correcting the vast majority of damage before it can lead to permanent mutations.
Specialized Subversion of DNA Repair: Antibody Diversity in B-Lymphocytes
In certain biological contexts, repair mechanisms are intentionally modulated or subverted. A notable example is the generation of antibody diversity in B lymphocytes:
Activation-Induced Cytidine Deaminase (AID): In B lymphocytes, the enzyme AID introduces mutations in the variable regions of immunoglobulin genes. AID deaminates cytosine to uracil in .
Error-Prone Repair and Replication: The uracil bases are then processed by error-prone repair pathways or bypassed by low-fidelity DNA polymerases, leading to mutations in the antibody genes.
Increased Antibody Diversity: This process of somatic hypermutation increases the diversity of antibodies, enabling the immune system to recognize a broader range of antigens.
In this case, controlled mutagenesis is beneficial for generating diversity, contrasting with the general role of repair pathways in maintaining genome stability.
Nobel Prize for DNA Repair Discoveries
The significance of repair research was recognized with the 2015 Nobel Prize in Chemistry, awarded to:
Thomas Lindahl: For mechanistic studies of base excision repair and the discovery of glycosylases, such as Uracil-DNA Glycosylase (Ung), which removes uracil from . Lindahl’s work established the concept of inherent instability and the existence of active cellular repair systems.
Paul Modrich and Aziz Sancar: For their work on mismatch repair and nucleotide excision repair pathways, respectively, completing our understanding of major DNA repair mechanisms.
These discoveries have been foundational in our understanding of genome maintenance and its relevance to human health and disease.
Hydrogen Bonding and Complementary Base Pairing
Specificity of Watson-Crick Base Pairs: A-T and G-C
Specificity of Watson-Crick Base Pairs: A-T and G-C. Hydrogen bonding between nitrogenous bases is the primary force stabilizing the double-helical structure of and defining specific base pairing. The canonical Watson-Crick base pairs are:
Adenine (A) and Thymine (T) in DNA, or Adenine (A) and Uracil (U) in RNA: Formed by two hydrogen bonds. One hydrogen bond is between the amino group at position 6 of adenine (donor) and the keto group at position 4 of thymine/uracil (acceptor). The second is between the nitrogen at position 1 of adenine (acceptor) and the imino group at position 3 of thymine/uracil (donor).
Guanine (G) and Cytosine (C): Formed by three hydrogen bonds, providing greater stability compared to A-T/A-U pairs. These include hydrogen bonds between the keto group at position 6 of guanine (acceptor) and the amino group at position 4 of cytosine (donor), the nitrogen at position 1 of guanine (donor) and the keto group at position 2 of cytosine (acceptor), and the amino group at position 2 of guanine (donor) and the nitrogen at position 3 of cytosine (acceptor).
This specificity arises from the precise complementarity of hydrogen bond donors and acceptors on the interacting faces of each base pair.
Stability and Denaturation of the DNA Double Helix
Hydrogen bonds, along with base stacking interactions, contribute to the stability of the double helix. However, hydrogen bonds are relatively weak non-covalent interactions, which allows for:
DNA Denaturation (Melting): The double helix can be disrupted, or denatured, by breaking the hydrogen bonds between base pairs. Denaturation can be induced by:
Increased Temperature: Heat increases the kinetic energy of molecules, disrupting hydrogen bonds.
Extreme pH: High or low pH conditions can alter the ionization state of bases, interfering with hydrogen bonding. For example, at pH 8 and above, strong bases can disrupt hydrogen bonds.
Reversibility of Denaturation (Renaturation or Annealing): denaturation is typically reversible. Upon removal of the denaturing agent (e.g., cooling down a heated solution or neutralizing pH), the complementary strands can re-associate and reform the double helix through renaturation or annealing.
DNA Elasticity: The ability to transiently break and reform hydrogen bonds makes molecule elastic. This elasticity is crucial for processes like replication and transcription.
DNA Elasticity in Biological Processes
elasticity, the capacity for reversible strand separation, is essential for several biological processes:
DNA Replication and Transcription: Enzymes like polymerase and polymerase must access the genetic information encoded in . This requires local unwinding and separation of the double helix ahead of the enzyme. Hydrogen bonds are temporarily disrupted in the region being processed and reformed behind the enzyme as it moves along the .
DNA Repair: Many repair mechanisms require access to single-stranded or distortion of the double helix to recognize and repair lesions.
Risks of Denatured DNA and Cellular Protection
While elasticity is essential, denatured is also more vulnerable:
Increased Susceptibility to Damage: When is denatured, the bases are exposed to the cellular environment and become more susceptible to chemical modifications and damage, including reactions with water and ROS.
Cellular Protection Mechanisms: To minimize the risks associated with denatured , cells employ strategies to keep compacted and protected:
- DNA Binding Proteins: is associated with proteins, such as histones in eukaryotes and histone-like proteins in prokaryotes. These proteins help to compact into chromatin and nucleosomes, reducing exposure of bases.
Evolutionary Significance of DNA Stability and Gene Regulation
The balance between stability and the capacity for controlled, transient denaturation is critical for both genome maintenance and gene expression.
Gene Expression and Transcription: Genes that are actively transcribed require local denaturation of to allow access for polymerase. Highly transcribed genes may experience more frequent transient denaturation, potentially increasing their susceptibility to mutations over evolutionary time.
Genome Evolution: A moderately stable genome, with a controlled level of instability and repair, is essential for evolution. It allows for the accumulation of beneficial mutations that drive adaptation and diversification, while preventing excessive genomic instability that would be detrimental to cellular function.
Base Modifications, Tautomerism, and Mutagenesis
Enzymatic Base Modifications: Cytosine Methylation and Epigenetic Regulation
Nitrogenous bases can undergo enzymatic modifications, which play crucial roles in regulating gene expression and other cellular processes. A prominent example is cytosine methylation:
5-Methylcytosine (5mC) and 5-Hydroxymethylcytosine (5hmC): Cytosine can be enzymatically methylated atthe 5-position of the pyrimidine ring to form 5-methylcytosine (5mC). 5mC can be further modified to 5-hydroxymethylcytosine (5hmC).
Epigenetic Role in Gene Regulation: In eukaryotes, particularly in mammals, cytosine methylation, especially in CpG islands within gene promoters, is a major epigenetic modification. 5mC in promoter regions is typically associated with:
Chromatin Compaction: Methylation can lead to chromatin compaction and reduced accessibility of DNA to transcription factors.
Transcriptional Repression: Gene silencing or reduced gene expression.
Heritability of Methylation Patterns: DNA methylation patterns can be heritable through cell divisions, contributing to epigenetic inheritance.
Tautomeric Shifts: Keto-Enol and Amino-Imino Isomerization
Nitrogenous bases can exist in different tautomeric forms, which are structural isomers that differ in the position of a proton and a double bond. The most relevant tautomerism for nucleic acids involves:
Keto-Enol Tautomerism (for oxo bases like Guanine, Thymine, Uracil): The predominant form is the keto form. However, bases can transiently shift to the less stable enol form.
Amino-Imino Tautomerism (for amino bases like Adenine, Cytosine): The predominant form is the amino form. Bases can transiently shift to the less stable imino form.
At physiological pH, the keto and amino forms are highly favored, with equilibrium ratios of keto to enol and amino to imino forms typically around \(10^4:1\).
Tautomerism as a Source of Spontaneous Mutations
Tautomeric shifts, although rare, can have significant biological consequences because they alter the hydrogen bonding potential of bases:
Altered Base Pairing Specificity:
Imino form of Cytosine (C*): The imino tautomer of cytosine (C*) can pair with adenine (A) instead of guanine (G).
Enol form of Guanine (G*): The enol tautomer of guanine (G*) can pair with thymine (T) instead of cytosine (C).
Mutagenesis during DNA Replication: If a base is in its rare tautomeric form during replication, it can lead to mispairing and incorporation of an incorrect base in the newly synthesized strand. For example:
- A transient shift of cytosine to its imino form (C*) during replication, where C* in the template strand pairs with adenine, can lead to a C \(\rightarrow\) T transition mutation in subsequent replication cycles. Starting with a G-C pair, after replication involving the C* tautomer and subsequent replication of the mutated strand, the original G-C pair is replaced by an A-T pair.
Spontaneous Mutations: Tautomerism is considered a significant source of spontaneous mutations in cells.
Mismatch Repair (MMR) and Correction of Tautomeric Errors
Cells possess Mismatch Repair (MMR) pathways that are crucial for correcting errors arising from tautomerism and other replication errors. MMR systems:
Recognize Mismatched Base Pairs: MMR enzymes scan newly synthesized for mismatched base pairs, including those resulting from tautomeric shifts.
Excise and Repair Mismatches: Upon detecting a mismatch, MMR systems excise a segment of the newly synthesized strand containing the error and resynthesize the region using the template strand as a guide, thus correcting the mismatch and maintaining genomic fidelity.
Effects of Ionization and pH on Base Properties
The ionization state of nitrogenous bases, and consequently their hydrogen bonding properties and the stability of nucleic acid structures, are sensitive to changes in pH.
Protonation and Deprotonation: Nitrogenous bases contain atoms (especially nitrogen atoms) that can be protonated or deprotonated depending on the pH of the environment.
pH-Dependent Hydrogen Bonding: Changes in ionization state alter the hydrogen bond donor and acceptor properties of the bases. For example, protonation of adenine at N1 at low pH changes N1 from a hydrogen bond acceptor to a donor.
DNA Denaturation at Extreme pH: Extreme pH values (very acidic or very alkaline) can disrupt the normal hydrogen bonding patterns in , leading to DNA denaturation. This is because altered ionization states interfere with the specific base pairing interactions that stabilize the double helix.
Minor and Anomalous Nucleobases
Besides the five major nucleobases (A, G, C, T, U), nucleic acids, particularly , contain a variety of minor or anomalous nucleobases. These are typically modified forms of the major bases.
Modified Bases in RNA: Structural and Functional Diversity
Modified bases are more prevalent in than in and play diverse structural and functional roles, especially in transfer (tRNA) and ribosomal (rRNA). Common types of modifications include:
Methylation: Addition of methyl groups to bases (e.g., 5-methylcytosine in , N6-methyladenosine).
Deamination: Removal of amino groups (e.g., conversion of adenine to inosine).
Isomerization: Rearrangement of atoms within the base (e.g., formation of pseudouridine).
Reduction: Saturation of double bonds (e.g., dihydrouridine).
Bulky Modifications: Addition of complex chemical groups, leading to bulky bases like ribothymidine and isopentenyladenosine.
These modifications can:
Alter Base Pairing Properties: Modified bases can have different hydrogen bonding capabilities, allowing for non-canonical base pairings (wobble base pairing in tRNA).
Stabilize RNA Structure: Some modifications enhance folding and stability.
Facilitate RNA-Protein Interactions: Modified bases can be recognized by specific RNA-binding proteins.
Examples of Anomalous Bases: Inosine, Pseudouridine, and Dihydrouridine
Examples of important anomalous bases found in include:
Inosine (I):
Formation: Formed by the enzymatic deamination of adenine. The corresponding nucleoside is inosine.
Base Pairing Properties: Inosine has a hypoxanthine base and can base pair with uracil (U), cytosine (C), and adenine (A). This wobble base pairing is crucial in tRNA, allowing a single tRNA to recognize multiple codons.
Pseudouridine (\(\Psi\)):
Isomer of Uridine: Pseudouridine (\(\Psi\)) is a C-glycoside isomer of uridine. In \(\Psi\), uracil is attached to ribose via a carbon-carbon (C5-C1’) bond instead of the normal N-glycosidic bond (N1-C1’).
Stabilizes RNA Structure: \(\Psi\) is thought to enhance structure stability, possibly through improved base stacking and hydrogen bonding.
Dihydrouridine (D):
Reduced Uracil: Dihydrouridine (D) is formed by the reduction of uracil, saturating the 5-6 double bond in the pyrimidine ring.
Structural Impact: D alters the local conformation of the and may contribute to flexibility.
These anomalous bases, particularly in tRNA, contribute to the complex three-dimensional structure and functional versatility of molecules by enabling diverse base pairing schemes beyond canonical Watson-Crick pairs.