DNA & RNA Replication

A · DNA Replication

• Semi-conservative (Meselson-Stahl, 1958): each daughter molecule has one parent strand + one new strand.
• Origin of replication opens a replication bubble with two replication forks moving outward.
• DNA polymerase always synthesizes 5′ → 3′, reading the template 3′ → 5′.

Enzyme cast list (memorize for FRQs)

Enzyme	Role
Helicase	Unwinds the double helix at the replication fork.
Topoisomerase (gyrase)	Relieves supercoiling ahead of the fork by transiently cutting and rejoining DNA.
Single-strand binding proteins (SSBs)	Bind separated strands to keep them apart.
Primase	Synthesizes a short RNA primer so DNA polymerase has a 3′-OH to extend from.
DNA polymerase III (prokaryotes)	Main replicative polymerase. Extends the primer 5′ → 3′. Eukaryotes use pols α/δ/ε.
DNA polymerase I (prokaryotes)	Removes the RNA primer and fills the gap with DNA.
DNA ligase	Seals nicks between Okazaki fragments — the final glue.
Telomerase (eukaryotes)	Extends telomere repeats in germline and stem cells.

Leading vs. lagging strand

• Leading strand: synthesized continuously toward the fork. One primer.
• Lagging strand: synthesized discontinuously away from the fork as Okazaki fragments, each requiring its own primer. Pol I removes primers; ligase seals.

Replication fork (ASCII)

     ←───── leading strand (continuous, toward fork)
        5′ ────────────────────►
   [helicase opens here →
        3′ ─────────── ◀──── 5′
                ◀──── 5′
            ◀────── 5′       ← lagging strand
                                 (Okazaki fragments)

B · Transcription (DNA → RNA)

• Occurs in the nucleus (eukaryotes) or cytoplasm (prokaryotes).
• RNA polymerase binds the promoter (TATA box in eukaryotes); transcription factors required for assembly.
• Reads template strand 3′ → 5′; synthesizes mRNA 5′ → 3′. Uses ribonucleotides (U replaces T).
• Three stages: initiation, elongation, termination.

mRNA processing (eukaryotes only)

1. 5′ cap — modified guanine. Protects mRNA and helps the ribosome bind.
2. 3′ poly-A tail — ~50–250 adenines. Stabilizes the mRNA and aids nuclear export.
3. Splicing — the spliceosome (snRNP complex) removes introns and joins exons.
4. Alternative splicing → multiple mature mRNAs from one pre-mRNA → multiple proteins from one gene. Major source of eukaryotic proteome diversity.

C · Translation (mRNA → Protein)

• At the ribosome, in the cytoplasm (or rough ER for membrane / secreted proteins).
• Genetic code: triplet, redundant, nearly universal, non-overlapping.
• Start codon: AUG (Met). Stop codons: UAA, UAG, UGA.
• tRNA brings amino acids; the anticodon pairs with the mRNA codon.

Ribosome sites

• A site — Aminoacyl tRNA arrives.
• P site — Peptidyl tRNA holds the growing chain.
• E site — Empty tRNA exits.

Three stages

1. Initiation: small ribosomal subunit + initiator tRNA (Met) find AUG; large subunit joins.
2. Elongation: tRNA enters A site → peptide bond forms (catalyzed by rRNA — a ribozyme) → ribosome translocates → empty tRNA exits via E.
3. Termination: stop codon recruits release factor → polypeptide released.

Post-translational modifications

Folding (chaperones), proteolytic cleavage, glycosylation, phosphorylation, signal-peptide-mediated targeting to organelles.

Prokaryote vs. eukaryote

Feature	Prokaryote	Eukaryote
Location of transcription & translation	Cytoplasm — simultaneous	Nucleus → cytoplasm — separate
mRNA processing	None (mostly)	5′ cap, poly-A, splicing
mRNA layout	Polycistronic (operons)	Monocistronic
Ribosome	70S	80S (cytoplasmic) / 70S (mito, chloroplast)

Example questions

MCQ Which enzyme joins Okazaki fragments? (A) Helicase (B) Primase (C) DNA polymerase III (D) DNA ligase

Answer: D. Ligase forms phosphodiester bonds, sealing the nicks between adjacent Okazaki fragments after Pol I has replaced their RNA primers with DNA.

FRQ Describe the role of mRNA processing in eukaryotic gene expression and explain how alternative splicing increases proteome diversity.

Answer: Eukaryotic pre-mRNA must undergo three modifications before translation: a 5′ G-cap is added to protect the mRNA and aid ribosome binding; a 3′ poly-A tail is added to stabilize it and assist export from the nucleus; and the spliceosome removes introns and joins exons to produce a mature mRNA. In alternative splicing, different combinations of exons from the same pre-mRNA are retained, so a single gene produces several mature mRNAs and therefore several distinct proteins. This expands the number of proteins encoded by the genome far beyond the gene count, contributing to eukaryotic complexity.

MCQ On the lagging strand, DNA is synthesized: (A) Continuously toward the fork (B) Continuously away from the fork (C) Discontinuously as Okazaki fragments (D) Only in eukaryotes

Answer: C. Because DNA polymerase only synthesizes 5′→3′, the strand running antiparallel to fork direction must be made in short Okazaki fragments, each restarted with its own primer.

FRQ A point mutation deletes one nucleotide in the middle of a coding sequence. Predict the consequence for the protein.

Answer: A single-nucleotide deletion causes a frameshift mutation. Every codon downstream of the deletion is read in a new reading frame, generally producing a completely different amino-acid sequence and often introducing a premature stop codon. The resulting protein is typically truncated, misfolded, and nonfunctional — or absent altogether.

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