The Dna

Structure Of Polynucleotide Chain

DNA, the genetic material in most organisms, is a long polymer of deoxyribonucleotides. Each nucleotide consists of a nitrogenous base (Purines: Adenine, Guanine; Pyrimidines: Cytosine, Thymine), a pentose sugar (deoxyribose), and a phosphate group. A nitrogenous base links to the 1' carbon of the sugar via a N-glycosidic linkage, forming a nucleoside. A phosphate group attaches to the 5' carbon of the sugar via a phosphoester linkage, forming a nucleotide. Nucleotides are linked by 3'-5' phosphodiester linkages to form a polynucleotide chain, with a free phosphate at the 5'-end and a free hydroxyl group at the 3'-end. RNA differs from DNA by having ribose sugar (with an extra -OH group at 2' position) and Uracil (U) instead of Thymine (T).

The DNA structure was proposed as a double helix by Watson and Crick in 1953, based on X-ray diffraction data. Key features include:

• Two polynucleotide chains with sugar-phosphate backbones and bases projecting inwards.
• Antiparallel polarity: One chain runs 5' to 3', the other 3' to 5'.
• Complementary base pairing: Adenine (A) pairs with Thymine (T) via two hydrogen bonds, and Guanine (G) pairs with Cytosine (C) via three hydrogen bonds. This purine-pyrimidine pairing maintains a uniform distance between the strands.
• The helix is right-handed, with a pitch of 3.4 nm and about 10 base pairs per turn, resulting in a distance of 0.34 nm between base pairs.
• Base stacking contributes to the stability of the helix.

The complementary nature of the strands allows for a simple copying mechanism (replication), where each strand acts as a template for synthesizing a new strand, leading to semiconservative replication.

Packaging Of Dna Helix

The immense length of DNA (about 2.2 meters in a mammalian cell) requires efficient packaging within the nucleus. In prokaryotes (like E. coli), DNA is organized into loops held by proteins in a region called the nucleoid. In eukaryotes, DNA is wrapped around positively charged histone proteins to form nucleosomes. A nucleosome consists of a histone octamer (8 histone molecules) around which approximately 200 base pairs of DNA are wrapped. These nucleosomes form 'beads-on-string' structures, which further condense into chromatin fibers, and finally into chromosomes during metaphase. Non-histone chromosomal proteins (NHC) are also involved in higher-level packaging. Regions of chromatin that are loosely packed (euchromatin) are transcriptionally active, while densely packed regions (heterochromatin) are inactive.

The Search For Genetic Material

The quest to identify the genetic material spanned decades. Early experiments by Frederick Griffith in 1928, using Streptococcus pneumoniae, demonstrated a 'transforming principle' that could convert harmless R-strain bacteria into virulent S-strain bacteria. However, the biochemical nature of this principle remained unknown.

The Genetic Material Is Dna

Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-1944) purified biochemical components from heat-killed S-strain bacteria. They found that DNA alone could transform R-strain bacteria into S-strain. Proteases and RNases did not inhibit transformation, but DNase did, strongly suggesting DNA as the transforming substance. However, not all scientists were convinced. The definitive proof came from Alfred Hershey and Martha Chase (1952) using bacteriophages. They labeled viral DNA with radioactive phosphorus (32P) and viral proteins with radioactive sulfur (35S). When phages infected bacteria, only the radioactive DNA entered the bacterial cell and was replicated, while the radioactive protein remained outside. This experiment unequivocally established DNA as the genetic material.

Properties Of Genetic Material (Dna Versus Rna)

A molecule acting as genetic material must be able to:

• Replicate itself.
• Be chemically and structurally stable.
• Allow for slow changes (mutations) for evolution.
• Express Mendelian traits.

Both DNA and RNA can replicate due to base pairing complementarity. However, DNA is generally preferred as genetic material because:

• Stability: DNA is chemically less reactive and structurally more stable than RNA. The 2'-OH group in RNA makes it more labile and prone to degradation.
• Repair: DNA's double-stranded structure with complementary strands allows for efficient repair mechanisms, further enhancing stability. The presence of thymine instead of uracil also adds stability.
• Reactivity: RNA's reactivity (being catalytic) makes it less suitable for long-term storage of genetic information.

While RNA can directly code for proteins, DNA relies on RNA for this process. Mutations occur in both, but RNA mutates faster due to its instability, making RNA viruses evolve more rapidly.

Rna World

The 'RNA world' hypothesis suggests that RNA was the first genetic material. It likely served as both the genetic information carrier and a catalyst (ribozyme) for essential early life processes like metabolism and translation. Later, DNA evolved from RNA, offering greater stability for genetic information storage, while RNA retained its roles in protein synthesis and other functions.

Replication

Watson and Crick proposed that DNA replicates semiconservatively, where each parental strand serves as a template for the synthesis of a new complementary strand. This mechanism was experimentally confirmed by Matthew Meselson and Franklin Stahl in 1958 using E. coli and 15N (heavy isotope of nitrogen). They observed that after one generation in a 14N medium, the DNA was hybrid (intermediate density), and after the second generation, it was a mixture of hybrid and light DNA, supporting the semiconservative model.

The Experimental Proof

Meselson and Stahl grew E. coli in a 15NH4Cl medium for many generations, incorporating 15N into their DNA. They then transferred these bacteria to a 14NH4Cl medium. DNA samples were extracted at intervals and analyzed using cesium chloride (CsCl) density gradient centrifugation. After 20 minutes (one generation), DNA was hybrid (15N-14N). After 40 minutes (second generation), it consisted of equal amounts of hybrid (15N-14N) and light (14N-14N) DNA, confirming semiconservative replication.

The Machinery And The Enzymes

DNA replication is a complex enzymatic process. The primary enzyme is DNA-dependent DNA polymerase, which synthesizes new DNA strands using a template. This enzyme is highly efficient and accurate, polymerizing about 2000 base pairs per second in E. coli. Deoxyribonucleoside triphosphates (dNTPs) act as substrates and provide energy. Other enzymes are crucial for the process. DNA replication occurs at a small unwound region called the replication fork. DNA polymerase synthesizes new strands only in the 5'→3' direction. This leads to continuous synthesis on the template strand with 3'→5' polarity (leading strand) and discontinuous synthesis on the template strand with 5'→3' polarity (lagging strand), forming Okazaki fragments. These fragments are later joined by DNA ligase. DNA replication requires an origin of replication (ori) to initiate. In eukaryotes, replication occurs during the S-phase of the cell cycle.

Transcription

Transcription is the process of synthesizing an RNA molecule from a DNA template, copying the genetic information. This process follows the principle of complementarity, with Adenine pairing with Uracil (instead of Thymine) and Guanine with Cytosine.

Transcription Unit

A transcription unit is a segment of DNA that is transcribed into RNA. It comprises three regions:

• Promoter: Located upstream (towards the 5' end) of the structural gene, it serves as the binding site for RNA polymerase and defines the start of transcription.
• Structural Gene: The DNA segment that codes for the RNA molecule. In eukaryotes, structural genes are often split into exons (coding sequences) and introns (intervening sequences).
• Terminator: Located downstream (towards the 3' end) of the coding strand, it signals the end of transcription.

The strand with 3'→5' polarity acts as the template strand, while the strand with 5'→3' polarity (having a sequence similar to RNA, with T replaced by U) is called the coding strand.

Transcription Unit And The Gene

A gene is defined as the functional unit of inheritance. In eukaryotes, genes are often split into exons and introns. The exons are expressed sequences that appear in the mature mRNA after splicing, while introns are removed. Regulatory sequences like promoters also influence gene expression and are sometimes considered regulatory genes.

Types Of Rna And The Process Of Transcription

In bacteria, three main types of RNA are transcribed: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). A single DNA-dependent RNA polymerase synthesizes all these RNA types. The process involves initiation (binding to the promoter), elongation (synthesis of RNA chain), and termination (release of RNA and polymerase). Transcription and translation can be coupled in bacteria because they occur in the same cellular compartment.

In eukaryotes, transcription is more complex:

• There are three nuclear RNA polymerases: RNA polymerase I (for rRNA), RNA polymerase III (for tRNA, 5S rRNA, snRNAs), and RNA polymerase II (for precursor of mRNA, hnRNA).
• The primary transcript (hnRNA) undergoes processing:
• Splicing: Introns are removed, and exons are joined.
• Capping: An unusual nucleotide (methyl guanosine triphosphate) is added to the 5' end.
• Tailing: Adenylate residues are added to the 3' end.
The processed hnRNA becomes functional mRNA and is transported to the cytoplasm for translation.

Genetic Code

The genetic code is a set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences). It's based on codons, triplet sequences of nucleotides in mRNA that specify a particular amino acid.

Discovery: George Gamow proposed that the code must be triplet (43 = 64 possible codons) to specify the 20 amino acids. Har Gobind Khorana and Marshall Nirenberg, using biochemical methods and cell-free systems, deciphered the genetic code.

Salient Features:

• Triplet: Codons are three nucleotides long.
• Degenerate: Most amino acids are coded by more than one codon.
• Unambiguous: Each codon codes for only one amino acid.
• Comma-less: Codons are read contiguously without punctuation.
• Universal: The code is nearly the same across all organisms, with minor exceptions in mitochondria and some protozoans.
• Initiator Codon: AUG serves as the start codon, initiating translation and also coding for Methionine.
• Stop Codons: UAA, UAG, and UGA signal the termination of translation.

Mutations And Genetic Code

Mutations, especially point mutations (single base changes) and frameshift mutations (insertions/deletions), can alter the genetic code. For example, a frameshift mutation in the beta-globin gene can change an amino acid (glutamic acid to valine), leading to sickle-cell anemia. Inserting or deleting one or two bases shifts the reading frame, altering the entire amino acid sequence downstream. Inserting or deleting three bases (or multiples thereof) adds or removes amino acids but maintains the reading frame.

Trna– The Adapter Molecule

Transfer RNA (tRNA) acts as an adapter molecule, bridging the mRNA codon and the corresponding amino acid. Each tRNA molecule has an anticodon loop that is complementary to an mRNA codon and an amino acid acceptor end where the specific amino acid binds. This binding is facilitated by aminoacyl-tRNA synthetases, a process called tRNA charging or aminoacylation. There are specific tRNAs for each amino acid, and an initiator tRNA for the start codon.

Translation

Translation is the process of synthesizing a polypeptide chain from the mRNA sequence. This occurs on ribosomes, the cellular machinery for protein synthesis. The process involves:

1. Initiation: The small ribosomal subunit binds to the mRNA at the start codon (AUG). The initiator tRNA, carrying Methionine, binds to the start codon.
2. Elongation: The ribosome moves along the mRNA, codon by codon. Charged tRNAs bind to the appropriate codons through anticodon-mRNA base pairing. The large ribosomal subunit facilitates the formation of peptide bonds between adjacent amino acids, with rRNA acting as a catalyst (ribozyme).
3. Termination: When the ribosome reaches a stop codon (UAA, UAG, UGA), a release factor binds, terminating translation and releasing the completed polypeptide chain.

Untranslated regions (UTRs) at the 5' and 3' ends of mRNA enhance the efficiency of translation.

Regulation Of Gene Expression

Gene expression, the process of producing a functional protein from a gene, is tightly regulated. In eukaryotes, regulation can occur at multiple levels: transcriptional initiation, RNA processing (splicing, capping, tailing), mRNA transport, and translational control. In prokaryotes, the rate of transcriptional initiation is the primary control point. This regulation is often achieved through the interaction of accessory proteins (activators and repressors) with promoter and operator regions of DNA.

The Lac Operon

The lac operon in E. coli is a classic example of prokaryotic gene regulation. It consists of a regulatory gene (i) and three structural genes (lacZ, lacY, lacA) that code for enzymes involved in lactose metabolism. The 'i' gene codes for a repressor protein that binds to the operator region, blocking RNA polymerase and preventing transcription. Lactose acts as an inducer; it binds to the repressor, inactivating it and allowing RNA polymerase to access the promoter, thereby initiating transcription of the structural genes. This system allows bacteria to efficiently utilize lactose as an energy source only when it is present in the environment.

Human Genome Project

The Human Genome Project (HGP), launched in 1990 and completed in 2003, was a monumental international effort to sequence the entire human genome, comprising approximately 3 billion base pairs. Its goals included identifying all human genes, determining the complete DNA sequence, storing information in databases, improving data analysis tools, developing technologies, and addressing ethical, legal, and social issues (ELSI).

Salient Features Of Human Genome

Key findings from the HGP include:

• Human genome has about 3164.7 million base pairs.
• The average gene size is around 3000 bases, but varies greatly.
• Estimated number of genes is 30,000–35,000 (lower than initially predicted).
• Over 50% of discovered genes have unknown functions.
• Less than 2% of the genome codes for proteins.
• Repetitive sequences constitute a large portion of the genome, thought to play roles in chromosome structure and evolution.
• Chromosome 1 has the most genes (approx. 2968), while the Y chromosome has the fewest (approx. 231).
• About 1.4 million single nucleotide polymorphisms (SNPs) have been identified, providing insights into human variation and disease association.

Applications And Future Challenges

The HGP has revolutionized biological research by enabling a genomics-based approach, allowing scientists to study genes and biological systems on a large scale. It has led to advancements in diagnosing, treating, and potentially preventing diseases. Future challenges involve interpreting the vast amount of data to understand biological systems fully and applying this knowledge to solve problems in healthcare, agriculture, and environmental remediation.

Dna Fingerprinting

DNA fingerprinting is a rapid technique used to identify differences in DNA sequences between individuals. It relies on the polymorphism (variations) found in repetitive DNA sequences, such as microsatellites and minisatellites (a type of satellite DNA called VNTRs – Variable Number of Tandem Repeats). These sequences, often non-coding, show high variability in copy number between individuals.

Technique: Developed by Alec Jeffreys, it typically involves DNA isolation, digestion with restriction enzymes, separation of fragments by electrophoresis, blotting onto membranes, hybridization with labeled VNTR probes, and detection by autoradiography. Modern techniques use PCR to amplify DNA from a single cell, increasing sensitivity.

Applications: DNA fingerprinting is a powerful tool in forensic science (identifying criminals), paternity testing, studying population diversity, and evolutionary biology. The unique banding pattern generated by VNTR analysis serves as an individual's genetic identity, except in cases of identical twins.

Polymorphism: Variations in DNA sequences, arising from mutations, are called polymorphisms. If an inheritable mutation occurs at a frequency greater than 0.01 in a population, it is considered a DNA polymorphism. These variations, especially in non-coding regions, are crucial for DNA fingerprinting and contribute to genetic diversity and evolution.

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