1. DNA Replication
DNA replication is the process by which a cell duplicates its genome before cell division. It is semi-conservative — each of the two resulting double helices contains one original (parental) strand and one newly synthesized daughter strand.
Replication Fork and Enzymes
Replication starts at specific sequences called origins of replication and proceeds bidirectionally, forming replication forks.
graph TD
A[Origin of Replication] --> B[Helicase unwinds DNA]
B --> C[Single-Strand Binding Proteins stabilize open strands]
B --> D[Topoisomerase relieves supercoiling strain]
C --> E[Primase synthesizes RNA Primers]
E --> F[DNA Polymerase III extends strands]
F -->|Continuous| G[Leading Strand: towards fork]
F -->|Discontinuous| H[Lagging Strand: Okazaki fragments]
H --> I[DNA Polymerase I replaces RNA with DNA]
I --> J[DNA Ligase seals nicks]
- DNA Helicase: Unwinds the double helix by breaking hydrogen bonds between base pairs, using ATP.
- Single-Strand Binding (SSB) Proteins: Bind to single-stranded DNA to prevent re-annealing or degradation.
- DNA Topoisomerase (Gyrase): Relieves structural strain/supercoiling ahead of the replication fork by making transient cuts in the sugar-phosphate backbone.
- DNA Primase: Synthesizes a short RNA primer (~10 nucleotides) providing a free 3'-OH group, which is required for DNA polymerases to initiate synthesis.
- DNA Polymerase III: The primary replicative enzyme in prokaryotes. It extends the new strand by adding deoxyribonucleoside triphosphates (dNTPs) to the 3'-OH end. Synthesis proceeds strictly in the 5' $\rightarrow$ 3' direction.
- Leading vs. Lagging Strand:
- The leading strand is synthesized continuously in the direction of fork movement.
- The lagging strand is synthesized discontinuously away from the fork as a series of Okazaki fragments.
- DNA Polymerase I: Removes the RNA primers via its 5' $\rightarrow$ 3' exonuclease activity and replaces them with DNA.
- DNA Ligase: Seals the nicks in the sugar-phosphate backbone by forming phosphodiester bonds, joining Okazaki fragments.
- Telomerase: A ribonucleoprotein that extends the 3' ends of eukaryotic chromosomes (telomeres) using an internal RNA template, preventing chromosome shortening during successive divisions.
2. Transcription (RNA Synthesis)
Transcription is the synthesis of an RNA molecule from a DNA template. It is catalyzed by RNA Polymerase.
Stages of Transcription
- Initiation: RNA Polymerase binds to a specific DNA region called the promoter upstream of the gene. In prokaryotes, this requires the sigma ($\sigma$) factor for promoter recognition. In eukaryotes, transcription factors bind to motifs like the TATA box to recruit RNA Polymerase II.
- Elongation: RNA Polymerase unwinds DNA and synthesizes RNA in the 5' $\rightarrow$ 3' direction, matching complementary RNA nucleotides (A, U, G, C) to the DNA template strand.
- Termination: In prokaryotes, termination is either intrinsic (hairpin loop in RNA destabilizes the enzyme) or Rho-dependent (Rho protein acts as a helicase to release the transcript). Eukaryotes terminate transcript synthesis after transcribing a polyadenylation signal sequence.
Post-Transcriptional Processing (Eukaryotes)
Eukaryotic primary transcripts (pre-mRNA) must undergo processing in the nucleus before transport to the cytoplasm:
- 5' Capping: Addition of a 7-methylguanosine cap to the 5' end to protect against nucleases and assist in ribosome binding.
- 3' Polyadenylation: Addition of a poly-A tail (100–250 adenine residues) to the 3' end by poly-A polymerase to stabilize the mRNA.
- Splicing: Spliceosomes remove non-coding introns and join coding exons together. Alternative splicing allows a single gene to encode multiple protein isoforms.
3. Translation (Protein Synthesis)
Translation is the process by which ribosomes decode the mRNA sequence to synthesize a polypeptide chain.
The Genetic Code
The genetic code is a triplet code: three nucleotides (a codon) specify one amino acid. The code is:
- Degenerate: Multiple codons can specify the same amino acid.
- Non-overlapping: Codons are read sequentially without gaps.
- Universal: The same codons specify the same amino acids in almost all organisms.
- Start Codon: AUG (methionine).
- Stop Codons: UAA, UAG, UGA (do not code for amino acids).
Components of Translation
- tRNA: Act as adaptors. Each tRNA has an anticodon complementary to an mRNA codon, and binds the corresponding amino acid at its 3' end.
- Aminoacyl-tRNA Synthetases: Catalyze the attachment of amino acids to their respective tRNAs (charging), utilizing ATP. This is a highly accurate two-step reaction.
- Ribosomes: Composed of a small subunit (decoding) and a large subunit (catalytic peptidyl transferase). Ribosomes contain three binding sites for tRNA:
- A (Aminoacyl) site: Binds incoming charged tRNA.
- P (Peptidyl) site: Holds the tRNA carrying the growing polypeptide chain.
- E (Exit) site: Holds uncharged tRNA before it leaves the ribosome.
graph LR
A[Ribosome A Site] -->|Translocation| B[Ribosome P Site]
B -->|Translocation| C[Ribosome E Site]
C -->|Exit| D[Uncharged tRNA released]
E[Incoming Aminoacyl-tRNA] --> A
Stages of Translation
- Initiation: The small ribosomal subunit binds mRNA (aided by the Shine-Dalgarno sequence in prokaryotes or the 5' cap in eukaryotes). The initiator tRNA ($tRNA_f^{Met}$ in prokaryotes; $tRNA^{Met}$ in eukaryotes) binds the AUG start codon at the P site, and the large subunit joins.
- Elongation:
- An aminoacyl-tRNA binds to the A site.
- Peptidyl Transferase (a ribozyme activity of the large subunit rRNA) catalyzes peptide bond formation, transferring the peptide chain from the P-site tRNA to the A-site amino acid.
- The ribosome translocates three nucleotides along mRNA (5' $\rightarrow$ 3'), moving the peptidyl-tRNA to the P site and the uncharged tRNA to the E site.
- Termination: When a stop codon enters the A site, Release Factors bind instead of a tRNA, triggering the hydrolysis of the peptide chain from the tRNA and disassembly of the ribosome.