Molecular Biology and Protein Synthesis

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The flow of genetic information: DNA → RNA → Protein. Replication: DNA is copied. Transcription: DNA → mRNA. Translation: mRNA → Protein. Exceptions: reverse transcription (retroviruses: RNA → DNA), RNA replication (RNA viruses). Formulated by Francis Crick (1958).

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A double helix of two antiparallel polynucleotide strands. Each nucleotide contains: Deoxyribose sugar + Phosphate group + Nitrogenous base (Adenine, Thymine, Guanine, Cytosine). Strands joined by hydrogen bonds between complementary base pairs: A-T (2 H-bonds), G-C (3 H-bonds).

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In any DNA molecule: [A] = [T] and [G] = [C] (A pairs with T; G pairs with C). Therefore: %A = %T and %G = %C. Implies the two strands are complementary. Evidence that led to the double helix model.

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Each new DNA molecule consists of one original (parental) strand and one newly synthesized strand. Proven by the Meselson-Stahl experiment (1958) using ¹⁵N/¹⁴N isotopes. The original strands serve as templates.

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Helicase: Unwinds and separates the double helix (breaks H-bonds). DNA Primase: Adds RNA primers (starting point). DNA Polymerase III: Synthesizes new DNA (5'→3' direction only). DNA Polymerase I: Replaces RNA primers with DNA. DNA Ligase: Joins Okazaki fragments and seals nicks.

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Leading strand: Synthesized continuously in the 5'→3' direction toward the replication fork. Lagging strand: Synthesized discontinuously in fragments (Okazaki fragments) away from the fork (also 5'→3') — then joined by DNA ligase. Results from the antiparallel nature of DNA and the inability of DNA polymerase to work 3'→5'.

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The process of synthesizing mRNA from a DNA template in the nucleus. Steps: Initiation (RNA polymerase binds promoter → DNA unwinds). Elongation (RNA polymerase adds complementary RNA nucleotides 5'→3'). Termination (reaches terminator sequence → mRNA released). Template strand read 3'→5'; mRNA synthesized 5'→3'.

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After transcription, pre-mRNA undergoes: (1) 5' capping (modified guanine cap — protects mRNA, aids ribosome binding). (2) Poly-A tail (100–200 adenines added to 3' end — stability, export from nucleus). (3) Splicing (introns removed, exons joined by spliceosomes). Mature mRNA is then exported to the cytoplasm.

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Introns (intervening sequences): Non-coding sequences in pre-mRNA that are removed (spliced out) before translation. Exons (expressed sequences): Coding sequences that are kept and joined to form the mature mRNA. Exons can be alternatively spliced — one gene can produce multiple proteins (alternative splicing).

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The synthesis of a polypeptide (protein) from mRNA sequence at the ribosome (in the cytoplasm for cytoplasmic proteins; at rough ER for secreted/membrane proteins). Requires mRNA, ribosomes, tRNA, amino acids, and initiation/elongation/release factors.

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A codon is a sequence of three mRNA nucleotides (triplet) that codes for one amino acid. The genetic code maps all 64 possible codons to 20 amino acids + 3 stop codons (UAA, UAG, UGA). The genetic code is: universal (same in all life), degenerate/redundant (multiple codons → same amino acid), non-overlapping.

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Transfer RNA — a clover-leaf shaped RNA molecule with: Anticodon (3 bases complementary to mRNA codon) at one end and the corresponding amino acid attached at the 3' end. tRNA brings the correct amino acid to the ribosome based on codon-anticodon pairing.

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Initiation: Ribosome assembles on mRNA at start codon (AUG — methionine); initiator tRNA binds. Elongation: Ribosome moves along mRNA codon by codon; tRNAs bring amino acids; peptide bonds form (peptidyl transferase). Termination: Stop codon reached (UAA, UAG, UGA); no tRNA matches; release factor → polypeptide released.

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A change in a single base pair in DNA. Substitution: One base replaced by another. Silent mutation: No change in amino acid (due to degeneracy of code). Missense mutation: Different amino acid incorporated. Nonsense mutation: Creates a premature stop codon → truncated protein.

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Insertion or deletion of one or more bases (not in multiples of 3) — shifts the reading frame of all downstream codons. Usually causes a completely different and non-functional protein. Often more severe than point mutations. Example: sickle cell anaemia is a point mutation; Tay-Sachs can result from frameshift.

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Caused by a single point mutation (missense) in the β-globin gene: adenine → thymine at position 6 → glutamic acid (hydrophilic) replaced by valine (hydrophobic). Mutant haemoglobin (HbS) polymerizes under low oxygen → red blood cells become rigid, sickle-shaped → blocked capillaries, haemolytic anaemia.

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A prokaryotic gene regulation system. In the absence of lactose: Repressor protein binds the operator → RNA polymerase blocked → lacZ, lacY, lacA genes NOT transcribed. In the presence of lactose: Lactose-derived allolactose binds and inactivates the repressor → operator free → genes transcribed → lactose-digesting enzymes produced.

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The control of when, where, and how much a gene is expressed. Not all genes are expressed in all cells at all times. Gene regulation explains cell differentiation (all cells have the same DNA but liver cells ≠ muscle cells), response to environment, and development.

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Promoter: DNA sequence where RNA polymerase binds to initiate transcription (upstream of the gene). Enhancer: DNA sequence that can be far from the gene (even thousands of base pairs away) — binds activator proteins → loops around to contact the promoter → greatly increases transcription rate.

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Changes in gene expression that do NOT involve changes to the DNA sequence itself, but can be heritable. Mechanisms: DNA methylation (usually silences genes), histone modification (acetylation/methylation of histone proteins → changes chromatin structure → affects transcription). Influenced by environment, diet, and experience.

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A technique to amplify (copy) a specific DNA sequence millions of times in vitro. Developed by Kary Mullis (Nobel Prize 1993). Steps: (1) Denaturation (~95°C — DNA strands separate). (2) Annealing (~55–65°C — primers bind). (3) Extension (~72°C — Taq polymerase synthesizes new DNA). Repeated ~30–40 cycles. Applications: forensics, diagnosis, research.

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A technique that separates DNA, RNA, or proteins by size using an electric field through a gel (agarose for nucleic acids). Smaller fragments move faster → travel further. Creates a band pattern used in DNA fingerprinting, diagnosing genetic diseases, and confirming PCR products.

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A revolutionary gene editing tool (Doudna & Charpentier — Nobel Prize 2020). Guide RNA directs Cas9 nuclease to a specific DNA sequence → Cas9 cuts both strands of DNA. The cell repairs the break: NHEJ (error-prone — disrupts gene) or HDR (precise editing using a template). Applications: treating genetic diseases, cancer, agriculture.

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The combination of DNA from two different sources (often different species) to create a novel DNA sequence. Process: (1) Cut DNA with restriction enzymes. (2) Insert into a vector (plasmid or virus). (3) Transform into a host cell. (4) Host cell expresses the gene. Applications: insulin production (from bacteria), vaccines, gene therapy, GMO crops.

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A natural gene-silencing mechanism. Small interfering RNA (siRNA) or microRNA (miRNA) — short double-stranded RNA — enters a cell → incorporated into the RISC complex → guided to complementary mRNA → mRNA degraded or translation blocked → gene expression reduced. Used as a research tool and emerging therapeutic approach.