DNA and Protein Synthesis

DNA Replication Basics

Key Points

  • Initiation at the origin of replication with helicase and primase.

  • Polymerase III extends RNA primers to form new strands.

  • Leading strand synthesis is continuous toward the replication fork.

  • Lagging strand synthesis produces Okazaki fragments, replaced by DNA polymerase I and sealed by ligase.

Initiation of DNA Replication

Origin of replication – specific DNA sequence where replication begins.

  • Helicase unwinds the double‑stranded DNA helix.

  • Single‑strand binding proteins (SSBs) bind to the exposed single strands, preventing re‑annealing.

  • Primase (an RNA polymerase) synthesizes a short RNA primer (~10 nt) complementary to the template strand.

RNA primer – short RNA segment that provides a 3′‑OH group for DNA polymerase to extend.

DNA Polymerase III: Main Replicative Enzyme

  • Adds deoxyribonucleotides only to the 3′‑phosphate (3′) end of an existing strand.

  • Cannot initiate synthesis; requires a primer.

DNA polymerase III – the primary enzyme that elongates the new DNA strand by incorporating deoxyribonucleotides.

Leading Strand Synthesis

  • Oriented toward the replication fork.

  • Continuous synthesis because the template strand runs 3′→5′, allowing polymerase to add nucleotides in the 5′→3′ direction without interruption.

Key Points

  1. Primase lays down a single RNA primer at the origin.

  2. DNA polymerase III extends the primer continuously.

  3. No additional primers are needed until the fork progresses.

Lagging Strand Synthesis

  • Oriented away from the replication fork.

  • Synthesized discontinuously as Okazaki fragments.

Process Overview

  1. Primase adds an RNA primer near the replication fork.

  2. DNA polymerase III extends this primer, forming an Okazaki fragment.

  3. Upon reaching the 5′ RNA primer of the previous fragment, DNA polymerase III detaches.

  4. DNA polymerase I replaces the RNA primer with DNA, removing RNA nucleotides with its 5′→3′ exonuclease activity and filling the gap with deoxyribonucleotides.

  5. DNA ligase seals adjacent DNA fragments by forming phosphodiester bonds.

  6. The cycle repeats as the fork advances, producing a series of fragments that are later joined.

Enzyme Coordination on Lagging Strand

Enzyme

Primary Function

Sequence of Action

Primase

Synthesizes RNA primer

Step 1

DNA polymerase III

Extends primer to form Okazaki fragment

Step 2

DNA polymerase I

Removes RNA primer, replaces with DNA

Step 4

DNA ligase

Joins DNA fragments (phosphodiester bond)

Step 5

Structural Insight into DNA Polymerase III

  • Historically depicted as two separate units (one for each strand).

  • Current model: two subunits function together, allowing the enzyme complex to replicate both strands simultaneously.

  • The lagging‑strand template folds, enabling the dimeric DNA polymerase III to access both leading and lagging strands without dissociating.

Dimeric DNA polymerase III – a single enzyme complex with two active sites that coordinates synthesis on both strands.

  • DNA Replication

    • Starts at origin of replication (eukaryotes have multiple origin of replication - linear DNA and prokaryotes have only one - circular DNA)

    • Helicase: breaks hydrogen bonds/separates strands

    • Topoisomerase: relieve stress on the DNA by helicase (prevents supercoiling)

    • Single Strand Binding Proteins: ensure strands stay separated

    • Primase: adds RNA primer to site of replication

    • DNA polymerase 3: add nucleotides from 5’ —> 3’ to elongate strand

    • DNA polymerase 1: destroys RNA primer and adds correct nucleotides

    • Ligase: joins the strand created by DNA polymerase 3 with DNA polymerase 1

Protein synthesis lecture (video lecture)

Overview

  • Protein synthesis is the process by which cells make proteins.

  • It occurs in all living cells and is essential for cell function.

  • Divided into two main steps:

    1. Transcription → DNA → mRNA

    2. Translation → mRNA → Protein

I. Transcription

Purpose

  • Converts DNA sequence → mRNA sequence (same “nucleic acid language”).

  • Occurs in the nucleus of eukaryotic cells.

Main Enzyme

  • RNA polymerase – reads the DNA and synthesizes mRNA.

Steps of Transcription

  1. Initiation

    • RNA polymerase binds to the promoter region on DNA (before the start site).

    • In prokaryotes: promoter at –35 and –10 regions.

    • In eukaryotes: promoter at –75 and –25 regions (e.g., TATA box).

    • The transcription starts at the +1 site.

  2. Elongation

    • RNA polymerase moves along the template strand, adding RNA nucleotides.

    • Base-pairing rule: A–U, T–A, G–C, C–G.

    • The mRNA sequence is complementary to the template strand and identical to the coding strand (except U replaces T).

  3. Termination

    • Transcription ends when RNA polymerase reaches a termination signal (region rich in A–T pairs → weaker bonds → polymerase detaches).

II. Post-Transcriptional Modifications (Eukaryotes Only)

After transcription, mRNA must be modified before leaving the nucleus.

1. G-Cap (5′ cap)

  • Added to the 5′ end.

  • Function:

    • Protects mRNA from degradation.

    • Helps ribosome recognize and bind to mRNA.

    • Aids in export from the nucleus.

2. Poly-A Tail

  • Added to the 3′ end.

  • Function:

    • Protects mRNA from degradation.

    • Aids in transport and ribosome binding.

3. RNA Splicing

  • Introns (noncoding regions) are removed.

  • Exons (coding regions) are joined.

  • Alternative splicing allows one gene to code for multiple proteins.

    • Example: Different combinations of exons → different protein variants.

III. Translation

Purpose

  • Converts mRNA sequence → amino acid sequence (protein).

  • Occurs in the cytoplasm (for both eukaryotes and prokaryotes).

Key Players

  • mRNA – carries the genetic code.

  • tRNA – brings amino acids; has anticodons complementary to mRNA codons.

  • rRNA + proteins – make up the ribosome (large + small subunits).

Steps of Translation

  1. Initiation

    • Ribosome binds to mRNA (at the G-cap in eukaryotes).

    • Scans until it finds start codon AUG, which codes for methionine.

  2. Elongation

    • Ribosome has three sites:

      • A site (Addition): tRNA enters carrying an amino acid.

      • P site (Peptide): peptide bond forms between amino acids.

      • E site (Exit): tRNA leaves after transferring its amino acid.

    • The ribosome moves along mRNA, adding amino acids one by one → forming a polypeptide chain.

  3. Termination

    • When ribosome reaches a stop codon (UAA, UAG, or UGA):

      • No tRNA binds.

      • A release factor binds instead.

      • The ribosome and mRNA disassemble, and the new protein is released.

IV. Post-Translational Modifications

  • Occurs after translation, especially for proteins leaving the cell.

  • Modifications can include:

    • Folding

    • Addition of chemical groups

    • Cleavage into smaller units

V. Summary Table

Step

Location

Key Enzymes / Molecules

Start Signal

End Signal

Product

Transcription

Nucleus

RNA Polymerase

Promoter

Terminator

mRNA

Post-Transcription

Nucleus

Spliceosome, enzymes

—

—

Mature mRNA

Translation

Cytoplasm

Ribosome (rRNA + proteins), tRNA

Start codon (AUG)

Stop codon (UAA, UAG, UGA)

Polypeptide (protein)

VI. Key Concepts to Remember

  • Transcription and translation both have initiation, elongation, and termination steps.

  • Coding strand and mRNA share the same sequence (except T → U).

  • Alternative splicing = more protein variety from fewer genes.

  • AUG = start, UAA, UAG, UGA = stop.

  • Post-transcriptional = modify mRNA.

  • Post-translational = modify protein.

Mutations

1. Point Mutations

  • Definition: Occur when only one base pair is changed.

  • Types:

    • Substitution – one base is replaced by another.

Effects of Point Mutations

  • Silent Effect:

    • Example: CCU → CCG (both code for Proline).

    • Due to the wobble effect — there are 64 codon combinations but only 20 amino acids.

    • No change in the amino acid sequence.

  • Missense Effect:

    • Changes one amino acid to another.

    • Type 1 (Minimal): The new amino acid has similar properties (e.g., nonpolar → nonpolar).

    • Type 2 (Dramatic): The new amino acid has different properties (e.g., nonpolar → polar), which can affect protein function.

  • Nonsense Effect:

    • A codon changes to a stop codon (or a stop codon changes to an amino acid).

    • Results in premature termination or extended protein chains.

2. Frameshift Mutations

  • Definition: Occur when a base is inserted or deleted, shifting the reading frame.

  • Types:

    • Insertion: Adding one or more bases.

    • Deletion: Removing one or more bases.

Effects of Frameshift Mutations

  • The entire sequence of amino acids after the mutation is read incorrectly.

  • Can result in missense or nonsense outcomes.

  • Silent effects are rare because the reading frame changes drastically.