MOLECULAR BASIS OF INHERITANCE

DNA STRUCTURE

The fundamental molecule of heredity is Deoxyribonucleic Acid (DNA). Understanding its structure is key to understanding how genetic information is stored, replicated, and passed down through generations. The structure of DNA was famously elucidated by James Watson and Francis Crick in 1953, building upon the work of Rosalind Franklin and Maurice Wilkins.

Components of DNA

DNA is a polymer, meaning it is made up of repeating subunits called nucleotides. Each nucleotide consists of three parts:

1. A deoxyribose sugar (a five-carbon sugar).
2. A phosphate group.
3. A nitrogenous base.

These three components together form a deoxyribonucleotide. When multiple deoxyribonucleotides link together, they form a DNA strand.

Nitrogenous Bases

There are four types of nitrogenous bases found in DNA:

• Purines: Adenine (A) and Guanine (G). These have a double-ring structure.
• Pyrimidines: Cytosine (C) and Thymine (T). These have a single-ring structure.

The DNA Polymer

Nucleotides are linked together by phosphodiester bonds. The phosphate group of one nucleotide forms a bond with the 3' carbon of the deoxyribose sugar of the next nucleotide. This creates a sugar-phosphate backbone for the DNA strand. Each strand has a directionality, with a free 5' phosphate group at one end and a free 3' hydroxyl group at the other. This is referred to as the 5' to 3' directionality.

The Double Helix Model

Watson and Crick proposed that DNA exists as a double helix. This means there are two polynucleotide strands coiled around a central axis. The key features of this model are:

• Antiparallel Strands: The two strands run in opposite directions. One strand runs from 5' to 3', and the complementary strand runs from 3' to 5'.
• Complementary Base Pairing: The nitrogenous bases on one strand pair with specific bases on the other strand. Adenine (A) always pairs with Thymine (T) via two hydrogen bonds, and Guanine (G) always pairs with Cytosine (C) via three hydrogen bonds. This is known as Chargaff's rule.
• Helix Dimensions: The double helix has a diameter of about 2 nanometers (20 Å). The distance between adjacent base pairs is about 0.34 nanometers (3.4 Å). One complete turn of the helix spans about 3.4 nanometers (34 Å) and contains approximately 10 base pairs.
• Major and Minor Grooves: The coiling of the strands creates two grooves on the surface of the helix: a wider major groove and a narrower minor groove. These grooves are important for the binding of proteins that interact with DNA.

Significance of DNA Structure

The double helix structure of DNA is perfectly suited for its functions:

• Information Storage: The sequence of bases along the DNA strand encodes genetic information.
• Replication: The complementary base pairing allows each strand to serve as a template for the synthesis of a new complementary strand, ensuring accurate duplication of genetic material.
• Mutation: Changes in the base sequence can lead to mutations, which are the source of genetic variation.

DNA REPLICATION

DNA replication is the process by which a cell makes an identical copy of its DNA. This is a crucial step before cell division (mitosis and meiosis) to ensure that each daughter cell receives a complete set of genetic instructions. The process is described as semi-conservative.

Semi-Conservative Replication

In semi-conservative replication, each new DNA molecule consists of one original (parental) strand and one newly synthesized strand. This model was experimentally confirmed by Meselson and Stahl.

Steps of DNA Replication

Replication is a complex process involving numerous enzymes and proteins. The key steps are:

1. Initiation: Replication begins at specific sites on the DNA called origins of replication (ori). Proteins bind to these sites and unwind the DNA.
2. Unwinding: The enzyme helicase unwinds the DNA double helix by breaking the hydrogen bonds between complementary bases. This creates a Y-shaped structure called a replication fork. Single-strand binding proteins (SSBs) bind to the separated strands to prevent them from re-annealing. Topoisomerase (or DNA gyrase in bacteria) relieves the torsional strain caused by unwinding.
3. Primer Synthesis: DNA polymerase cannot initiate DNA synthesis on its own. It requires a pre-existing 3'-OH group. An enzyme called primase synthesizes short RNA primers (about 5-10 nucleotides long) that are complementary to the template DNA strand. These primers provide the necessary 3'-OH group for DNA polymerase to start adding nucleotides.
4. Elongation: DNA polymerase III (in E. coli) is the main enzyme responsible for synthesizing new DNA strands. It reads the template strand in the 3' to 5' direction and synthesizes the new strand in the 5' to 3' direction by adding deoxyribonucleotides that are complementary to the template. The energy for this process comes from the hydrolysis of high-energy phosphate bonds in the incoming deoxyribonucleoside triphosphates (dNTPs).
5. Leading and Lagging Strands: Because DNA polymerase can only synthesize DNA in the 5' to 3' direction, replication occurs differently on the two template strands at the replication fork.
   • Leading Strand: The template strand that runs 3' to 5' allows for continuous synthesis of the new strand in the 5' to 3' direction. This new strand is called the leading strand.
   • Lagging Strand: The template strand that runs 5' to 3' requires discontinuous synthesis. DNA polymerase synthesizes short fragments of DNA called Okazaki fragments, each initiated by an RNA primer. These fragments are synthesized in the 5' to 3' direction, but overall movement is away from the replication fork.
6. Primer Removal and Ligation: After the Okazaki fragments are synthesized, the RNA primers are removed by DNA polymerase I (in E. coli). DNA polymerase I also fills in the gaps with DNA nucleotides. Finally, the enzyme DNA ligase joins the Okazaki fragments together by forming phosphodiester bonds, creating a continuous DNA strand.
7. Termination: Replication continues until the entire DNA molecule is copied. In circular bacterial chromosomes, replication terminates when the two replication forks meet. In linear eukaryotic chromosomes, replication continues until the ends of the chromosomes (telomeres) are reached.

Proofreading and Repair

DNA polymerases have a proofreading activity (3' to 5' exonuclease activity) that allows them to remove incorrectly incorporated nucleotides during replication. This significantly reduces the error rate. Cells also have complex DNA repair mechanisms to fix errors that escape proofreading.

TRANSCRIPTION

Transcription is the process of synthesizing an RNA molecule from a DNA template. It is the first step in gene expression, where the genetic information encoded in DNA is transcribed into a messenger RNA (mRNA) molecule.

The Central Dogma

The central dogma of molecular biology, as proposed by Francis Crick, states that genetic information flows from DNA to RNA to protein. Transcription is the DNA to RNA step. DNA $\rightarrow$ RNA $\rightarrow$ Protein

Enzyme Involved: RNA Polymerase

The primary enzyme responsible for transcription is RNA polymerase. In eukaryotes, there are multiple types of RNA polymerases, each transcribing different types of RNA. In prokaryotes, there is usually a single type of RNA polymerase. RNA polymerase reads the DNA template strand in the 3' to 5' direction and synthesizes an RNA molecule in the 5' to 3' direction, using ribonucleoside triphosphates (ATP, UTP, CTP, GTP) as substrates. Unlike DNA polymerases, RNA polymerase does not require a primer to initiate synthesis.

Stages of Transcription

Transcription occurs in three main stages:

1. Initiation:
   • Transcription begins when RNA polymerase binds to a specific region on the DNA called the promoter. The promoter is a DNA sequence located upstream of the gene that signals the starting point for transcription.
   • In prokaryotes, a sigma (σ) factor subunit of RNA polymerase recognizes and binds to the promoter.
   • In eukaryotes, transcription initiation is more complex, involving various transcription factors that bind to the promoter and recruit RNA polymerase.
   • Once bound, RNA polymerase unwinds a small portion of the DNA double helix, creating a transcription bubble.
2. Elongation:
   • RNA polymerase moves along the DNA template strand in the 3' to 5' direction.
   • As it moves, it synthesizes a complementary RNA strand by adding ribonucleotides to the 3' end of the growing RNA chain. The base pairing rules are A with U (uracil replaces thymine in RNA) and G with C.
   • The DNA double helix re-forms behind the polymerase as it moves forward.
3. Termination:
   • Transcription stops when RNA polymerase encounters a terminator sequence on the DNA.
   • In prokaryotes, termination can occur via two main mechanisms:
     • Rho-independent (intrinsic) termination: The terminator sequence forms a hairpin loop structure in the RNA, followed by a string of uracils. This structure causes the RNA polymerase to pause and detach from the DNA.
     • Rho-dependent termination: A protein called Rho factor binds to the nascent RNA and moves towards the RNA polymerase. When the polymerase pauses at the terminator sequence, Rho catches up and causes the dissociation of the RNA polymerase and the RNA transcript from the DNA.
   • In eukaryotes, termination mechanisms are more varied and depend on the specific RNA polymerase and the type of RNA being transcribed. For mRNA, termination is coupled with RNA processing events.

RNA Processing in Eukaryotes

In eukaryotes, the primary transcript produced by RNA polymerase (pre-mRNA) undergoes several processing steps in the nucleus before it can be translated in the cytoplasm. These steps include:

• Capping: A modified guanine nucleotide (7-methylguanosine cap) is added to the 5' end of the pre-mRNA. This cap protects the mRNA from degradation and is important for ribosome binding during translation.
• Tailing: A string of adenine nucleotides (poly-A tail), typically 50-250 bases long, is added to the 3' end of the pre-mRNA. The poly-A tail enhances mRNA stability, aids in its export from the nucleus, and plays a role in translation.
• Splicing: Eukaryotic genes contain non-coding regions called introns, interspersed among coding regions called exons. During splicing, introns are removed from the pre-mRNA, and the exons are joined together to form a mature mRNA molecule. This process is carried out by a complex of proteins and small nuclear RNAs (snRNAs) called the spliceosome.

GENETIC CODE

The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells. It is read as a sequence of nucleotide triplets called codons.

Characteristics of the Genetic Code

The genetic code has several important characteristics:

• Triplet Code: The code is read in groups of three nucleotides. Each triplet, or codon, specifies a particular amino acid or a signal for termination.
• Degenerate/Redundant: Most amino acids are specified by more than one codon. For example, leucine is specified by six different codons. This degeneracy helps to minimize the impact of mutations.
• Unambiguous: Each codon specifies only one amino acid (or a stop signal). There is no ambiguity in the code.
• Universal: The genetic code is virtually universal across all living organisms, from bacteria to humans. The same codons specify the same amino acids in most cases. Minor exceptions exist, such as in mitochondria and some protozoa.
• Commaless: The code is read continuously without any punctuation or gaps between codons.
• Initiation Codon: The codon AUG typically serves as the start codon, initiating translation and also coding for the amino acid methionine.
• Termination Codons: Three codons (UAA, UAG, UGA) act as stop codons, signaling the termination of translation. They do not code for any amino acid.

The Codon Table

The genetic code can be represented in a codon table, which shows the amino acid specified by each of the 64 possible codons. The table is read by looking at the first base in the row, the second base in the column, and the third base within the corresponding box.

1st Base	2nd Base				3rd Base
	U	C	A	G
U	Phe (UUU, UUC)				Leu (UUA, UUG)	U	Cys (UGU, UGC)	Trp (UGG)	U	Ser (UCU, UCC, UCA, UCG)
	Ser (UUC, UUC)	U	Stop (UAA, UAG)	Gly (UGG)
	C	Leu (CUU, CUC, CUA, CUG)				Pro (CCU, CCC, CCA, CCG)	U	His (CAU, CAC)	Gln (CAA, CAG)	C	Ser (AGU, AGC)
		Ile (AUU, AUC, AUA)	Met (AUG) / Start	Thr (ACU, ACC, ACA, ACG)	Ala (GCU, GCC, GCA, GCG)							A	Asn (AAU, AAC)	Lys (AAA, AAG)	A	Asp (GAU, GAC)	Glu (GAA, GAG)	G
A		Val (GUU, GUC, GUA, GUG)				U	Arg (CGU, CGC, CGA, CGG)	C	Stop (UGA)	Trp (UGG)	G	Ser (AGU, AGC)
		Tyr (UAU, UAC)	Stop (UAA)	Cys (UGU)	Trp (UGG)
	G	Ala (GCU, GCC, GCA, GCG)				Asp (GAU, GAC)	Glu (GAA, GAG)	U	Arg (CGU, CGC, CGA, CGG)	C	Ser (AGU, AGC)
		Asn (AAU, AAC)	Lys (AAA, AAG)	A	His (CAU, CAC)							Gln (CAA, CAG)	G

Translation: From Genetic Code to Protein

Translation is the process by which the genetic information in mRNA is used to synthesize a specific sequence of amino acids, forming a protein. This process occurs in the ribosomes in the cytoplasm.

Key Players in Translation

• mRNA: Carries the genetic code from DNA to the ribosome.
• Ribosomes: The cellular machinery responsible for protein synthesis. They consist of ribosomal RNA (rRNA) and proteins. Ribosomes have two subunits (large and small) and binding sites for mRNA and tRNAs.
• tRNA (transfer RNA): Acts as an adapter molecule. Each tRNA molecule has an anticodon loop that is complementary to a specific mRNA codon, and it carries the corresponding amino acid at its other end.

Steps of Translation

Translation also proceeds in three main stages:

1. Initiation:
   • The small ribosomal subunit binds to the mRNA, usually near the 5' end, and scans for the start codon (AUG).
   • The initiator tRNA, carrying methionine, binds to the start codon.
   • The large ribosomal subunit then joins the complex, positioning the initiator tRNA in the P (peptidyl) site.
2. Elongation:
   • A tRNA carrying the next amino acid (specified by the next codon on the mRNA) enters the A (aminoacyl) site of the ribosome.
   • A peptide bond is formed between the amino acid on the tRNA in the P site and the amino acid on the tRNA in the A site. This reaction is catalyzed by peptidyl transferase activity of the large ribosomal subunit.
   • The ribosome then translocates (moves) one codon down the mRNA in the 5' to 3' direction. The tRNA that was in the P site moves to the E (exit) site and is released, while the tRNA that was in the A site (now carrying the growing polypeptide chain) moves to the P site.
   • The A site is now free to accept the next incoming tRNA. This cycle repeats, adding amino acids to the polypeptide chain.
3. Termination:
   • When the ribosome encounters a stop codon (UAA, UAG, or UGA) in the A site, there is no tRNA that recognizes these codons.
   • Instead, release factors bind to the stop codon.
   • This binding triggers the hydrolysis of the bond between the polypeptide chain and the tRNA in the P site, releasing the completed protein.
   • The ribosomal subunits, mRNA, and release factors then dissociate.

Regulation of Gene Expression

While this section covers the core molecular mechanisms, it's important to note that gene expression is tightly regulated at various levels. This ensures that proteins are produced only when and where they are needed, conserving energy and maintaining cellular function. Regulation can occur during transcription initiation, RNA processing, mRNA stability, translation, or post-translational modification.

SUMMARY OF KEY CONCEPTS

The molecular basis of inheritance revolves around DNA structure, replication, transcription, and translation, all guided by the genetic code.

• DNA Structure: Double helix, antiparallel strands, complementary base pairing (A-T, G-C), sugar-phosphate backbone.
• DNA Replication: Semi-conservative process, involving helicase, primase, DNA polymerases, and ligase, ensuring accurate duplication of genetic material.
• Transcription: Synthesis of RNA from a DNA template by RNA polymerase, involving initiation, elongation, and termination. Eukaryotic mRNA undergoes capping, tailing, and splicing.
• Genetic Code: Triplet, degenerate, unambiguous, and nearly universal code that dictates the sequence of amino acids in proteins.
• Translation: Synthesis of proteins from mRNA templates by ribosomes, with the help of tRNA, involving initiation, elongation, and termination.