Lectures 5-7 Structure & Function of Nucleic acids V2

Chapter 3: Nucleic Acid Structure and Function

3.1 Structure of DNA & RNA

Nucleotides and Constituents

• Nucleotides are the basic building blocks of nucleic acids like DNA and RNA. Each nucleotide is composed of three distinct parts:

  • Phosphate group: This group contributes to the overall negative charge of the nucleic acid strand and links nucleotides together.

  • Sugar: The sugar component can be ribose (found in RNA) or deoxyribose (found in DNA), differing by the presence of an oxygen atom.

  • Nitrogenous base: There are two kinds of nitrogenous bases –

    • Purines: adenine (A), guanine (G)

      • two rings

      • found in both DNA and RNA

    • Pyrimidines: thymine (T) only found in DNA, cytosine (C), and Uracil (U) only found in RNA

      • one ring

Primary Structure of DNA

• The primary structure of DNA refers to the linear sequence of nucleotides connected by phosphodiester bonds. These bonds form between the phosphate group of one nucleotide and the sugar of another.

• DNA has directionality, with distinct 5' (five prime) and 3' (three prime) ends, which are important for processes like replication and transcription.

Secondary Structure of DNA

• The secondary structure of DNA is characterized by the formation of a double helix, where two strands of DNA are coiled around each other.

  • The strands run antiparallel, meaning one strand runs from 5' to 3' while the other runs from 3' to 5'.

  • Complementary base pairing occurs between the nitrogenous bases: adenine pairs with thymine (A-T) and guanine pairs with cytosine (G-C), stabilized by hydrogen bonds.

  • The Watson-Crick model describes this helical structure as having major and minor grooves, providing access for proteins to bind.

Antiparallel Nature

• Understanding the antiparallel nature of DNA is crucial for its replication and transcription, where enzymes read the template strand in a specific direction (3' to 5') to synthesize RNA in a 5' to 3' direction.

Coding vs. Template Strand

• Within a double-stranded DNA molecule, the coding strand is identical to the mRNA transcript (except for uracil replacing thymine). In contrast, the template strand is the one used during transcription to synthesize mRNA, providing the complementary sequence.

Chargaff's Rule

• Chargaff's rules states that in DNA, the amount of adenine equals thymine (A = T) and the amount of guanine equals cytosine (G = C). This foundational pairing is essential for maintaining the double helix's stability.

Types of DNA

• A-DNA: Shorter and wider than B-DNA, this form is typically found under dehydrated conditions and has 11 base pairs per turn.

• B-DNA: The most prevalent form of DNA in cells, characterized as a right-handed helix with about 10.5 base pairs per turn, forming a stable, relaxed structure.

• Z-DNA: A left-handed helix with a zig-zag backbone, which is less common but may play a role in gene regulation and is transiently formed under certain conditions.

Hyperchromic Effect

• The hyperchromic effect refers to the increase in UV absorbance of DNA as the double helix unwinds, which is helpful in determining the melting temperature (Tm) of DNA - the temperature at which half of the DNA strands are denatured.

Factors Impacting Melting Temperature (Tm)

• Several factors affect Tm, including:

  • Strand length: Longer strands have higher stability, requiring more heat to denature.

  • GC content: Higher content of guanine and cytosine, which form three hydrogen bonds as opposed to the two in A-T pairs, increases thermal stability.

  • Ionic strength: Increased ionic strength stabilizes the double helix by shielding negative charges on the phosphate backbone.

Supercoiling Types

• Positive supercoiling occurs when DNA is overwound, while negative supercoiling occurs when DNA is under-wound, the latter being a common feature in cellular DNA that helps facilitate replication and transcription.

Type I and Type II Isomerases

• Type I Isomerases: These enzymes cut one strand of the DNA, allowing for relaxation of supercoils.

• Type II Isomerases: These enzymes cut both strands of DNA and use ATP to introduce or remove supercoils, crucial for DNA replication and transcription processes.

Histone Proteins

• Histones play a vital role in the packaging of DNA into a more compact structure known as chromatin. They have a highly positive charge, allowing them to bind tightly to the negatively charged DNA.

• Histone binding is generally nonspecific compared to specific binding interactions, such as those between transcription factors and gene promoter regions, which require precise recognition and binding.

3.2 Genomics: The Study of Genomes

Definition of Genome

• A genome is defined as the complete set of genetic material within an organism, including both coding (genes) and non-coding sequences, which influence gene regulation and intergenic functions.

Human Genome Project

• The Human Genome Project was a landmark scientific endeavor that mapped and sequenced the entire human genome, consisting of approximately 3 billion base pairs and around 30,000 genes. This project has paved the way for advancements in personalized medicine and genomics research.

Euchromatin vs. Heterochromatin

• Euchromatin: Less densely packed form of chromatin, actively involved in transcription, making genes accessible for expression.

• Heterochromatin: More condensed and transcriptionally inert form, often found at centromeres and telomeres; plays roles in maintaining chromosome structure and stability.

Gene Structure

• Genes encompass two main components:

  • Exons: Coding sequences that are expressed in the final mRNA product.

  • Introns: Non-coding sequences that are removed during RNA splicing, essential for regulatory functions in post-transcriptional modifications.

Polymorphisms

• Genetic variation is often represented by single nucleotide polymorphisms (SNPs), which are common point mutations, and short tandem repeats (STRs), microsatellite sequences used in genetic profiling and forensic studies.

Genomic Studies

• Researchers use tools and databases such as BLAST for sequence comparison and analysis, enabling the identification of genes and evaluation of genetic variations across different species and individuals.

3.3 Methods in Nucleic Acid Biochemistry

Plasmids for Genetic Transfer

• Plasmids are small, circular DNA molecules that replicate independently of chromosomal DNA and are widely used in biotechnology for genetic engineering.

• Methods for genetic transfer include:

  • Conjugation: Direct transfer of plasmids between bacterial cells through direct contact.

  • Transduction: Transfer of genetic material via bacteriophages.

  • Transformation: Uptake of naked DNA from the environment into a bacterial cell.

Plasmids Description

• Plasmids serve as essential tools in molecular biology for cloning, gene expression, and gene therapy applications, functioning as vectors that can carry and propagate foreign DNA fragments.

Restriction Enzymes

• Restriction enzymes are proteins that can cut DNA at specific recognition sites, facilitating the manipulation of DNA fragments in recombinant DNA technology, cloning, and genetic analysis.

PCR Cycle Phases

• The Polymerase Chain Reaction (PCR) is a technique used to amplify specific DNA sequences. It involves three key phases:

  • Phase I: Denaturation at 95-98 ºC to separate DNA strands.

  • Phase II: Annealing at 50-60 ºC to allow primers to bind to the target DNA sequences.

  • Phase III: Extension at 68-72 ºC, where DNA polymerase synthesizes new strands by adding nucleotides.

cDNA Libraries

• cDNA libraries represent collections of complementary DNA (cDNA) generated from mRNA, allowing for the study of gene expression profiles. The process involves:

  • Reverse transcription of mRNA to convert it into cDNA.

  • Amplification of cDNA fragments.

  • Ligation of cDNA into plasmid vectors for cloning and analysis.

Sanger Sequencing

• Sanger sequencing, or chain termination method, utilizes dNTPs as normal building blocks and modified ddNTPs that terminate DNA strand elongation when incorporated. The process includes:

  • Incorporation of fluorescent markers on ddNTPs for rapid detection and automated sequencing, allowing for high-throughput sequencing applications.