Basic Molecular Biology Chapter-2

 

Central Dogma of Molecular Biology

The Central Dogma of Molecular Biology explains the flow of genetic information within a biological system. It is the process by which the information in genes flows into proteins: DNA → RNA → Protein.

Reference: https://www.researchgate.net/figure/Annotating-the-central-dogma-of-molecular-biology-An-illustrated-version-of-the-central_fig1_280631767

DNA Replication (for more details refer DNA (Deoxyribonucleic Acid) Chapter-5)

1. Mechanisms of Replication

   • Origin of Replication: The specific sequence at which replication begins. In prokaryotes, there is usually a single origin of replication, while eukaryotes have multiple origins.
   • Replication Fork: The Y-shaped structure formed during DNA replication where the DNA is split into two strands and replication occurs.
   • Leading and Lagging Strand Synthesis: DNA replication is semi-discontinuous. The leading strand is synthesized continuously, while the lagging strand is synthesized in short segments called Okazaki fragments.

2. Key Enzymes and Proteins

   • DNA Polymerases: Enzymes that synthesize new DNA strands by adding nucleotides to a pre-existing strand.
   • Helicase: Unwinds the DNA double helix at the replication fork.
   • Primase: Synthesizes RNA primers that provide a starting point for DNA polymerase.
   • Ligase: Joins Okazaki fragments on the lagging strand by forming phosphodiester bonds.
   • Single-Strand Binding Proteins (SSBs): Stabilize single-stranded DNA and prevent it from re-annealing or forming secondary structures.

3. Replication Models

• Semi-Conservative Model: Each of the two daughter DNA molecules contains one old strand and one newly synthesized strand.
• Conservative Model: The original DNA molecule is conserved, and a completely new molecule is synthesized.
• Dispersive Model: Parental and newly synthesized DNA segments are interspersed in both strands following replication.

1. Proofreading and Repair (for more details refer DNA (Deoxyribonucleic Acid) Chapter-6)

   • Proofreading Activity of DNA Polymerase: DNA polymerase has a 3' to 5' exonuclease activity that corrects errors during DNA synthesis.
   • Mismatch Repair: Corrects errors missed by proofreading.
   • Excision Repair: Removes and replaces damaged DNA, such as thymine dimers caused by UV light.

Transcription

1. RNA Synthesis

   • Initiation: RNA polymerase binds to the promoter region of DNA and begins RNA synthesis.
   • Elongation: RNA polymerase moves along the DNA template, synthesizing RNA in the 5' to 3' direction.
   • Termination: RNA synthesis stops when RNA polymerase reaches a termination signal in the DNA.

2. Types of RNA

   • mRNA (Messenger RNA): Carries the genetic code from DNA to the ribosome for protein synthesis.
   • tRNA (Transfer RNA): Brings amino acids to the ribosome during translation.
   • rRNA (Ribosomal RNA): Combines with proteins to form ribosomes.
   • snRNA (Small Nuclear RNA): Involved in mRNA splicing.
   • miRNA (Micro RNA): Regulates gene expression by interfering with mRNA.

3. Transcription in Prokaryotes vs Eukaryotes

   • Prokaryotes: Transcription and translation occur simultaneously in the cytoplasm. Sigma factors help RNA polymerase recognize the promoter.
   • Eukaryotes: Transcription occurs in the nucleus, and RNA undergoes extensive processing before translation. Transcription factors and RNA polymerase II are involved in initiation.

4. Post-Transcriptional Modifications

   • 5' Capping: Addition of a modified guanine nucleotide to the 5' end of the mRNA.
   • Polyadenylation: Addition of a poly(A) tail to the 3' end of the mRNA.
   • Splicing: Removal of introns and joining of exons to produce mature mRNA.
   • RNA Editing: Alteration of nucleotide sequences in RNA.

Translation

1. Mechanism of Translation

• Initiation: The small ribosomal subunit binds to the mRNA, and the initiator tRNA recognizes the start codon (AUG).
• Elongation: tRNAs bring amino acids to the ribosome, and peptide bonds form between amino acids.
• Termination: The ribosome reaches a stop codon, and the newly synthesized polypeptide is released.

       Reference: https://www.pinterest.com/pin/protein-synthesis-location-process-steps-diagram--750412356687646002/

1. Ribosome Structure and Function

   • Ribosomal Subunits: Composed of a small and a large subunit that come together during translation.
   • Sites within the Ribosome:
     • A (Aminoacyl) Site: Holds the incoming tRNA with the next amino acid.
     • P (Peptidyl) Site: Holds the tRNA with the growing polypeptide chain.
     • E (Exit) Site: Where the empty tRNA exits the ribosome.

2. tRNA and Aminoacyl-tRNA Synthetase

   • tRNA Structure: Cloverleaf structure with an anticodon at one end and an amino acid attachment site at the other.
   • Charging of tRNA: Aminoacyl-tRNA synthetase attaches the correct amino acid to its corresponding tRNA.

3. Genetic Code

   • Codon-Anticodon Pairing: The codon in mRNA pairs with the anticodon in tRNA.
   • Start and Stop Codons: Start codon (AUG) initiates translation, while stop codons (UAA, UAG, UGA) terminate it.
   • Wobble Hypothesis: The third base in a codon can pair with multiple bases in the anticodon, allowing for flexibility.

4. Post-Translational Modifications

   • Phosphorylation: Addition of phosphate groups.
   • Glycosylation: Addition of sugar moieties.
   • Proteolytic Cleavage: Removal of specific peptide segments to activate the protein.

Gene Regulation (related to Central Dogma)

1. Operon Model in Prokaryotes

   • Lac Operon: Regulates lactose metabolism. In the absence of lactose, the repressor binds to the operator, preventing transcription. In the presence of lactose, the repressor is inactivated, allowing transcription.
   • Trp Operon: Regulates tryptophan synthesis. When tryptophan is abundant, it binds to the repressor, activating it to inhibit transcription.

2. Transcriptional Regulation in Eukaryotes

   • Promoters, Enhancers, Silencers: DNA elements that regulate transcription.
   • Transcription Factors: Proteins that bind to DNA and influence RNA polymerase binding and activity.
   • Epigenetic Modifications:
     • DNA Methylation: Addition of methyl groups to DNA, usually suppressing gene expression.
     • Histone Modification: Addition or removal of chemical groups to histones, affecting chromatin structure and gene expression.

Key Experiments and Historical Context (for more details refer DNA (Deoxyribonucleic Acid) Chapter-3)

1. Griffith's Experiment (1928)

   • Demonstrated the "transforming principle" by showing that non-virulent bacteria could become virulent when mixed with heat-killed virulent bacteria.

2. Avery-MacLeod-McCarty Experiment (1944)

   • Identified DNA as the "transforming principle" by showing that DNA, not proteins or RNA, could transform bacteria.

3. Hershey-Chase Experiment (1952)

   • Confirmed that DNA is the genetic material by using bacteriophages labeled with radioactive isotopes.

4. Meselson-Stahl Experiment (1958)

• Provided evidence for the semi-conservative model of DNA replication using isotopic labeling and density gradient centrifugation.

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