Unit 3: DNA structure, replication, expression and regulation

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Mutation

Translation

Protein Transport

Unit 3: DNA structure, replication, expression and regulation

Translation & Protein Trafficking

Two types of Ribosomes

Bound Ribosomes

Free Ribsomes

Permanent change in DNA sequence

Types of Mutations

Frameshift Mutations

Reading frame shift

Deletion

Insertion

Point Mutations

Nonsense

Creates premature stop codon

Missense

Changes one amino acid

Silent

No change in amino acid

Secretory Pathway

Final vesicle sends protein to:

Cel Membrane

Lysosome

Plasma Membrane

Golgi further modifies, sorts, packages.

Travels to Golgi Apparatus in vesicles.

Protein enters ER lumen ).

Signal peptide directs ribosome to ER membrane.

Steps of Translation

3. Termination

Ribosomal subunits separate.

Release factor binds

Ribosome reaches a stop codon (UAA, UAG, UGA).

2. Elongation

Amino acids are joined by peptide bonds

Matching tRNAs bring amino acids.

Ribosome moves along mRNA, reading codons.

1. Initiation

Large ribosomal subunit joins to form complete ribosome.

tRNA carries methionine to start codon

Small ribosomal subunits bind to mRNA at start codon (AUG)

mRNA -> Protein

Prokaryotes

No mRNA processing

smaller ribosomes

Eukaryotes

larger ribosomes

mRNA Processing: Capped, spliced, poly-A-Tail

DNA Structure & Replication

DNA as Genetic Material

Chargaff's rule (A = T, C = G)

DNA vs. protein as genetic material

Hershey & Chase bacteriophage experiment

Griffith's transformation experiment

DNA Replication

Features of Replication

Polymerases need a primer to begin synthesis

Requires dNTPs as substrates

High fidelity (1 error per 10⁶ bases, proofreading)

High speed (E. coli: 2000 nt/sec)

Leading vs Lagging Strand

Directionality matters: DNA pol adds only to 3′ end

Okazaki fragments

Lagging strand synthesized discontinuously

Leading strand synthesized continuously

Overview

Replication bubble

Bidirectional replication

Replication fork

Replication origin (ORI)

Meselson-Stahl experiment (14N vs. 15N labeling)

Semiconservative replication model

Enzymes in DNA Replication

Ligase – seals nicks between Okazaki fragments

DNA Polymerase I – removes RNA primers & fills gaps

Sliding clamp – increases DNA pol processivity

DNA Polymerase III – synthesizes new strand (5′→3′)

Primase – adds RNA primers

Single-Stranded Binding Proteins (SSBPs) – stabilize separated strands

Topoisomerase – relieves tension ahead of fork

Helicase – unwinds DNA

DNA Structure

Hydrogen bonds (between bases)

Phosphodiester bonds (covalent, between nucleotides)

Complementary base pairing: A–T (2 H-bonds), G–C (3 H-bonds)

Nitrogenous bases: purines (A, G) vs pyrimidines (T, C)

Sugar-phosphate backbone

Antiparallel strands (5′→3′ / 3′→5′)

Double helix model (Watson & Crick)

Transcription & RNA Processing

Transcription

Eukaryotes

RNA polymerase detaches

Cleavage factors cut RNA transcript

RNA polymerase reads termination factor

U --> T in RNA

RNA polymerase reads 5' --> 3'

synthesizes complementary RNA 3' --> 5'

Initiation

forms transcription bubble

Transcription factors bind to promotor sequnce

recruits RNA Polymerase II to bind to promotor

Promotor region

Enhancer sequences

Promotor = TATA box

Location

goes to cytoplasm for translation

Nucleus

Prokaryotes

Termination

Newly synthesized mRNA released

Termination factor reached

Elongation

RNA synthesis in the 5' --> 3' direction

RNA polymerase adds complementary nucleotides

C-G

A-U

Initiaton

Topoisomerase keeps it from rewinding and relieves stress

Helicase unwinds DNA at promotor

Unwinds DNA

Forms transcription bubble

RNA polymerase binds to promotor

promotor = specific sequece on DNA

Location

Cytoplasm

Transcription and Translation coupled here

RNA processing

Splicing

Alternative splicing

makes many proteins from one gene

Splicesome

snRNA + proteins

Joins exons

Removes introns

3' Poly-A tail

Poly-A Polymerase adds A LOT of A's to 3' end

~100-300

Cut RNA downstream

Cleavage factor binds

5' Cap

Protects mRNA

Happens as RNA is synthesized

Eukaryotes only