Genetics of Microorganisms - Module 2: Transposition and Chromosome Structure

Transposition

  • Involves the integration of small DNA segments into new genomic locations.
  • Can occur at many different locations within the genome.
  • Transposable elements (TEs) are small, mobile DNA segments, sometimes called "jumping genes."
  • First identified by Barbara McClintock in corn during the early 1950s.
  • Found in diverse species: bacteria, fungi, plants, and animals.

Barbara McClintock

  • Awarded the Nobel Prize in Physiology or Medicine in 1983 for her discovery of mobile genetic elements.

Different Transposition Pathways

  • Transposable elements (TEs) move via different transposition pathways.
  • Two general types:
    • Simple transposition
    • Retrotransposition

Simple Transposition

  • Widely used by transposons in bacterial and eukaryotic species.
  • TE is removed from its original site and transferred to a new target site.
  • Mechanism: cut-and-paste.

Retrotransposition

  • Transposable elements (retrotransposons) move via an RNA intermediate.
  • Transcribed into RNA.
  • Found only in eukaryotic species.

Insertion Elements and Simple Transposons

  • All TEs are flanked by direct repeats (DRs): identical base sequences oriented in the same direction.
  • Simplest TE is an insertion element, flanked by inverted repeats.
  • Inverted repeats: identical (or very similar) DNA sequences running in opposite directions, ranging from 9 to 40 bp.
  • May contain a gene for transposase, which catalyzes transposition.

LTR Retrotransposons

  • Evolutionarily related to retroviruses.
  • Retain the ability to move around the genome but generally do not produce mature viral particles.
  • Contain long terminal repeats (LTRs) at both ends (typically a few hundred base pairs in length).
  • Code virally related proteins, such as reverse transcriptase and integrase, needed for retrotransposition.

Non-LTR Retrotransposons

  • Do not resemble retroviruses in having LTR sequences.
  • May contain a gene encoding a protein that functions as both a reverse transcriptase and an endonuclease.
  • Some are evolutionarily derived from normal eukaryotic genes.
  • Example: Alu family of repetitive sequences in humans, derived from the 7SL RNA gene. Approximately 1 million copies exist.

Transposase

  • Catalyzes the excision and insertion of TEs.
  • Recognizes inverted repeats at the ends of a TE, bringing them close together.
  • Transposase cleaves the target DNA at staggered sites.
  • The transposable element is inserted into the target site.
  • DNA gap repair synthesis results in direct repeats.

Simple Transposition and Copy Number

  • Transposition can increase copy number.
  • Occurs after the replication fork has passed through the TE, creating two copies.
  • One TE can transpose ahead of the fork, where it is copied again.
  • One chromosome retains one TE, while the other gains an additional copy.

Retrotransposons and Reverse Transcriptase

  • Retrotransposons use an RNA intermediate.
  • LTR retrotransposon movement requires reverse transcriptase (synthesizes double-stranded DNA from RNA template) and integrase (recognizes LTRs, cuts target site, and inserts TE).

Target-Site Primed Reverse Transcription

  • Non-LTR retrotransposons move via target-site primed reverse transcription.
  • Retrotransposon transcribed into RNA with a 3′ polyA tail.
  • Target DNA recognized by endonuclease.
  • PolyA tail binds to nicked site.
  • Reverse transcriptase uses target DNA as a primer to make a DNA copy of the RNA.

Transposable Elements: Mutation and Evolution

  • Occur in the genomes of all species.
  • Abundance varies across species:
    • Frog (Xenopus laevis): 77%
    • Corn (Zea mays): 60%
    • Human (Homo sapiens): 45%
    • Mouse (Mus musculus): 40%
    • Fruit fly (Drosophila melanogaster): 20%
    • Nematode (Caenorhabditis elegans): 12%
    • Yeast (Saccharomyces cerevisiae): 4%
    • Bacterium (Escherichia coli): 0.3%

Repetitive Sequences in Eukaryotic Genomes

  • Some repetitive sequences are due to TE proliferation.
  • In mammals:
    • LINEs (Long interspersed elements): 1,000 to 10,000 bp long, 20,000 to 1,000,000 copies per genome, ~17% of the human genome.
    • SINEs (Short interspersed elements): < 500 bp long. Example: Alu sequence (~1,000,000 copies in the human genome, 10% of the genome).

Biological Significance of Transposons

  • Evolutionary role debated.
    • Selfish DNA theory: TEs exist because they can proliferate without significantly harming the host.
    • TEs offer some advantage (e.g., bacterial TEs carrying antibiotic-resistance genes).
  • TEs may cause greater genetic variability through recombination.
  • TEs may cause exon shuffling (insertion of exons into coding sequences), leading to genes with more diverse functions.

Transposons and Rapid Genomic Change

  • Transposable elements can rapidly enter and proliferate in a genome.
  • Example: P element in Drosophila melanogaster, introduced around the 1950s, has since spread worldwide.
  • Transposable elements have a variety of effects on chromosome structure and gene expression.

Consequences of Transposition

  • Chromosome Structure
    • Chromosome breakage: Excision of a TE.
    • Chromosomal rearrangements: Homologous recombination between TEs at different locations.
  • Gene Expression
    • Mutation: Incorrect excision of TEs.
    • Gene inactivation: Insertion of a TE into a gene.
    • Alteration in gene regulation: Transposition of a gene next to regulatory sequences or vice versa.
    • Alteration in exon content: Insertion of exons into the coding sequence via TEs (exon shuffling).

Regulation of Transposition

  • Many outcomes are harmful, so transposition is highly regulated.
  • Occurs in few individuals under certain conditions.
  • Agents like radiation, chemical mutagens, and hormones can stimulate TE movement.

Structure of Eukaryotic Chromosomes in Nondividing Cells

  • Linear DNA must be tightly compacted to fit within the nucleus (2 to 4 µm in diameter), despite being over 1 meter long if stretched end to end.
  • Compaction involves interactions between DNA and proteins.
  • DNA-protein complex is called chromatin.
  • Proteins bound to DNA are subject to change, affecting chromatin compaction.

Nucleosomes

  • The repeating structural unit within eukaryotic chromatin.
  • Composed of a double-stranded DNA segment (146 bp) wrapped 1.65 times around a histone octamer.
  • Histone octamer: two copies each of four different histone proteins.

Histones

  • Histone proteins are basic (contain many positively charged amino acids like lysine and arginine).
  • Bind to negatively charged phosphates along the DNA backbone.
  • Have a globular domain and a flexible, charged amino terminus (tail).
  • Five types of histones:
    • H2A, H2B, H3, and H4 (core histones): Two of each form the octamer.
    • H1 (linker histone): Binds to DNA in the linker region, helps organize adjacent nucleosomes.

Nucleosome Organization

  • H1 histone not bound: beads on a string.
  • H1 histone bound to linker region: nucleosomes more compact.

Nucleosomes and Chromatin Structure

  • 30nm fiber model, depicted long-range interactions of nucleosomes to form a fiber; this model is no longer accepted
  • Nucleosomes associate with each other to form a more compact structure.
  • Histone H1 plays a role in this compaction.
  • Zigzag model: linker DNA is relatively straight, and nucleosomes form a zigzag arrangement (occurs over short distances).

Further Compaction

  • Chromatin can be further compacted into loop domains.
  • Loop extrusion model:
    • SMC proteins (structural maintenance of chromosomes): Form a dimer that wraps around two DNA segments to form a loop.
    • CCCTC binding factor (CTCF): Stabilizes loops by binding to DNA and then to each other.

Topologically Associated Domains (TADs)

  • Chromatin is organized into TADs (approximately 100 kb to 1 Mb in length).
  • DNA segments within a TAD are more likely to interact with each other.
  • TAD boundaries are determined by SMC proteins and CTCFs.
  • Promote interactions within a TAD and prevent interactions between different TADs (act as insulators).

Heterochromatin and Euchromatin

  • Heterochromatin: Tightly compacted regions of chromosomes, transcriptionally inactive.
  • Euchromatin: Less condensed regions of chromosomes, transcriptionally active.

Types of Heterochromatin

  • Constitutive heterochromatin: Always heterochromatic, permanently inactive, contains highly repetitive sequences.
  • Facultative heterochromatin: Can interconvert between euchromatin and heterochromatin.

Chromosome Territories

  • Each chromosome in the cell nucleus is found in a discrete chromosome territory.

Four-Level Hierarchy of Chromosome Organization

  • Level 1: Chromosomes occupy distinct territories; interchromosomal interactions play a role in chromosomal arrangements.
  • Level 2: Active genes associate with other active genes in euchromatin; repressed genes cluster together in heterochromatin.
  • Level 3: Chromosomes are organized into structural domains known as TADs (100 kb to 1 Mb).
  • Level 4: Chromosomal organization is largely determined by the affinity of nucleosomes for one another; short tri- or tetranucleosomes are arranged in a zigzag manner.

Chromosome Structure During Cell Division

  • During M phase, the level of compaction changes dramatically.
  • By the end of prophase, sister chromatids are entirely condensed.
  • Metaphase chromosomes undergo little gene transcription.

Metaphase Chromosomes

  • Chromatin within metaphase chromosomes is organized along a scaffold.
  • Scaffold contains SMC proteins.

Condensin I and II

  • Two multiprotein complexes that help form and organize metaphase chromosomes: Condensin I and Condensin II.
  • Play a critical role in chromosome condensation during M phase of mitosis and meiosis.
  • Both contain SMC proteins (structural maintenance of chromosomes).
  • SMC proteins use energy from ATP to catalyze loop formation.

Condensin Function

  • During early prophase, condensin II enters the nucleus and plays a role in condensation.
  • Condensin I binds to chromatin only after the nuclear envelope breaks apart.
  • After interphase, both condensin I and II facilitate the reorganization of chromosomes into radial loop arrays.
  • Condensin II forms a spiral scaffold and creates large loops attached to the scaffold in radial loop arrays.
  • Condensin I promotes the formation of smaller loops within the larger loops formed by Condensin II.

Cohesin

  • A multiprotein complex that also contains an SMC protein.
  • Found along the entire length of each chromatid until the middle of prophase.
  • Promotes binding between sister chromatids during mitosis and meiosis.
  • After the middle of prophase, cohesin is found only at the centromere.
  • Cohesin at the centromere is degraded at the start of anaphase.