Genes and Chromosomes

• Exons:

  • Coding segments of genes that are retained in mature mRNA
  • Directly encode amino acid sequences of proteins
  • Constitute only ~1.5% of the human genome
  • Are colinear with the polypeptide product they encode

• Introns:

  • Noncoding segments that interrupt the coding sequence
  • Are transcribed initially but removed during RNA splicing
  • Often much larger than exons (e.g., β-hemoglobin gene: one intron contains >50% of the gene's DNA)
  • Make up much of the ~28.5% of the human genome that consists of gene regions
  • Some genes have numerous introns (e.g., titin with 178 introns)
  • Few prokaryotic genes contain introns

Example: In the ovalbumin gene, seven introns interrupt eight exons, with the introns comprising 85% of the gene's DNA.

Key insight: The exon-intron structure allows for alternative splicing, creating multiple protein products from a single gene, but requires complex RNA processing machinery.

• Viruses:

  • Smallest genomes (4,000-200,000 base pairs)
  • May be DNA or RNA, single- or double-stranded
  • Often circular in replicative form
  • Tightly packed (DNA can be 290× longer than viral particle)
  • Example: Bacteriophage λ has 48,502 bp

• Bacteria:

  • Medium-sized genomes (~4-5 million bp)
  • Single, circular chromosome plus plasmids
  • Example: E. coli genome is 4,639,221 bp
  • Encodes ~4,300 protein genes and 115 RNA genes
  • DNA is ~850× longer than the cell

• Eukaryotes:

  • Large genomes (12 million to billions of bp)
  • Multiple linear chromosomes
  • Human: 3.2 billion bp across 46 chromosomes
  • Yeast: 12 million bp across 16 chromosomes
  • Most complex genomes have more non-coding than coding DNA

Key point: Genome size generally correlates with organism complexity, but the relationship is not strictly linear due to varying amounts of non-coding DNA.

The colinearity principle states that the nucleotide sequence in DNA directly corresponds to the amino acid sequence in the resulting protein through these relationships:

• DNA template strand (3'→5') produces complementary mRNA (5'→3') • mRNA codons (triplets of nucleotides) specify amino acids in the same sequential order • Each amino acid is determined by exactly three consecutive nucleotides (a codon) • The sequence progresses from the amino terminus to the carboxyl terminus of the polypeptide

Example: The DNA sequence 5'-ATGGCCGAT-3' would be transcribed to mRNA 5'-AUGGCCGAU-3', which would translate to the amino acid sequence Met-Ala-Asp.

Note: This principle applies to prokaryotes directly, while in eukaryotes it's complicated by the presence of introns that must be removed before translation.

Human somatic cells contain:

• 46 chromosomes (diploid number, 2n)
• 23 pairs of chromosomes
• 22 pairs of autosomes
• 1 pair of sex chromosomes (XX in females, XY in males)

In contrast, human gametes contain:

• 23 chromosomes (haploid number, n)
• No chromosome pairs (single copy of each)
• 22 autosomes
• 1 sex chromosome (X in eggs, X or Y in sperm)

This reduction in chromosome number occurs through meiosis and is essential for maintaining the correct chromosome number when gametes fuse during fertilization.

When DNA is underwound:

• The linking number (Lk) decreases from its relaxed value (Lk₀)
• The change is expressed as ΔLk = Lk - Lk₀, which is negative for underwound DNA
• This change is quantified by specific linking difference (σ): σ = ΔLk/Lk₀

For example, if a 2,100 bp circular DNA with Lk₀ = 200 is underwound by 2 turns:

• The new Lk becomes 198
• ΔLk = 198 - 200 = -2
• σ = -2/200 = -0.01, meaning 1% of helical turns have been removed

Cellular DNAs typically have σ values between -0.05 and -0.07, representing 5-7% underwinding.

• Negative supercoiling:

  • Results from underwinding the DNA double helix
  • Decreases the linking number (ΔLk is negative)
  • Example: Lk = 198 when Lk₀ = 200 (ΔLk = -2)
  • Forms right-handed supercoils
  • Biologically important: facilitates DNA processes requiring strand separation

• Positive supercoiling:

  • Results from overwinding the DNA double helix
  • Increases the linking number (ΔLk is positive)
  • Example: Lk = 202 when Lk₀ = 200 (ΔLk = +2)
  • Forms left-handed supercoils
  • Typically resolves through topoisomerase activity in cells

Relationship to topology:

• Underwinding reduces the linking number (Lk) of circular DNA below its relaxed value (Lk₀)
• This creates negative supercoiling where σ = (Lk - Lk₀)/Lk₀ (typically -0.05 to -0.07 in cells)
• The topological strain can be distributed between twist (local winding of the helix) and writhe (coiling of the helix axis)
• Underwound DNA cannot return to its relaxed state without strand breakage or topoisomerase action

Biological benefits:

• DNA replication: Facilitates local strand separation required for replication initiation
• Transcription: Aids RNA polymerase in unwinding the DNA at promoter regions
• Recombination: Promotes the formation of specialized structures (like cruciforms) that can serve as recognition sites
• DNA compaction: Contributes to DNA packaging through solenoidal supercoiling
• Gene regulation: Affects the binding affinity of regulatory proteins to specific DNA sequences
• DNA repair: Some repair processes require local strand separation to access damaged DNA

These benefits explain why cells actively maintain DNA in an underwound state through ATP-dependent processes, despite the energy cost involved.

The transient covalent protein-DNA linkage is crucial for topoisomerase I function because it:

1. Maintains the energy of the broken phosphodiester bond through the 5'-phospho-tyrosine link
2. Prevents complete dissociation of the DNA strands during strand passage
3. Enables controlled manipulation of DNA topology without risking permanent DNA damage
4. Provides a nucleophilic target (5'-phospho-tyrosine bond) for the 3'-hydroxyl group to attack during religation
5. Ensures topological changes occur without net energy input, as the energy stored in the protein-DNA linkage drives the religation step

This covalent intermediate is essential for the enzyme to change DNA linking number without requiring ATP hydrolysis, unlike type II topoisomerases.

• The tight wrapping of DNA around the histone core in nucleosomes removes about one helical turn in the DNA
• When a nucleosome binds to relaxed, closed-circular DNA:
  • The binding introduces a negative solenoidal supercoil where DNA wraps around the histone core
  • A compensatory positive supercoil forms in the unbound region (due to conservation of linking number)
  • Eukaryotic topoisomerases can relax the positive supercoil
  • This leaves the negative supercoil fixed through binding to the nucleosome
  • The net result is a decrease in linking number and underwound DNA

Contribution to DNA compaction hierarchy:

1. Nucleosomes (first level): 7-fold compaction of DNA
2. 30 nm fiber (second level): ~100-fold total compaction
3. Higher-order loops and scaffold attachments (next levels): up to 10,000-fold total compaction

Factors affecting 30 nm fiber formation:

• Histone H1 presence: Required for stability; regions being actively transcribed have reduced H1
• Histone modifications: Acetylation tends to disrupt 30 nm fiber formation
• Ionic conditions: Higher salt concentrations promote fiber formation
• Transcriptional activity: Actively transcribed regions exist in a less-ordered state
• Sequence-specific DNA-binding proteins: Can interrupt the regular pattern of the fiber
• ATP-dependent chromatin remodeling complexes: Can alter fiber structure

The 30 nm fiber represents a dynamic structure that can be modulated to regulate DNA accessibility for processes like transcription, replication, and repair.