Lecture 7: Mobile Genetic Elements and Transposition

Week 4 and Midterm Preparation

• This is week four, which means midterms are this week for some students. Ensure you are prepared by reviewing all lecture materials and notes.

• Our midterm is in week six. A practice exam is available. Take advantage of this to familiarize yourself with the format and types of questions.

• The midterm includes rapid-fire questions (50-60) and data interpretation questions (10). Prepare for both types of questions to maximize your score.

• Understand the relevance of molecules and how systems change if they don't work. Focus on how molecular malfunctions can lead to broader system failures within the body.

• Questions will focus on application, not just definitions. Be ready to apply your knowledge to solve problems and analyze scenarios.

• Utilize office hours, remote office line, email, and TAs for questions. Don't hesitate to seek clarification on any topics you find challenging.

Mobile Genetic Elements (Transposable Elements)

• Mobile genetic elements, also known as transposable elements or transposons, make up a significant portion of our genome. These elements can move within the genome, influencing gene expression and genome stability.

• These elements are being adapted for important processes like spermatogenesis. Research is ongoing to fully understand their role in this and other critical biological functions.

• The lecture focuses on the mechanisms of transposition. Pay close attention to the different ways these elements can move and insert themselves into new locations.

• Reading: Molecular Biology of the Cell (6th ed., pp. 287-292; 7th ed., pp. 306-317). Complement the lecture with the assigned reading to gain a deeper understanding of the material.

• Transposons: the mobile genetic elements themselves

• Transposition: the mechanism by which transposons move

• Transposase: an enzyme involved in transposition

Genome Size and the C-Value Enigma

• Scientists initially questioned if genome size predicts organism size. Early hypotheses suggested a direct correlation between genome size and organism complexity.

• Plants have diverse genomes (size ranging from 10^{4.8} to 10^8). This wide range in genome size among plants makes them excellent subjects for studying the factors influencing genome size.

• Birds and mammals have confined genomes. The relatively consistent genome sizes in these groups suggest evolutionary pressures that maintain a stable genome size.

• The C-value enigma: some organisms with large genomes are small, and vice versa. This paradox challenges simple assumptions about the relationship between genome size and organismal complexity.

The Human Genome Project and "Junk DNA"

• The Human Genome Project cost ilda $3,000,000,000 (approximately double that amount today due to inflation). This massive undertaking revolutionized our understanding of the human genome.

• It revealed approximately 22,000 genes in the human genome, fewer than expected. This discovery led to a re-evaluation of the role of non-coding DNA.

• The rest of the genome was initially termed "junk DNA." This term reflected the initial lack of understanding about the function of non-coding regions.

• Later research identified transposable elements as vestigial viruses within this "junk DNA." These elements are remnants of ancient viral infections that have become integrated into the host genome.

• These elements no longer form capsids or leave the cell like retroviruses. They have lost the ability to produce infectious viral particles but can still influence genome function.

Types of Transposable Elements

• Three main types:

  • DNA-only transposons

  • Retroviral-like retrotransposons

  • Non-retroviral retrotransposons

• Transposable elements can encode enzymes for removal, copying, and placement in the genome. These enzymes, such as transposase, are essential for the movement and integration of transposons.

• Some have lost the capacity to encode these enzymes or induce their expression. These non-autonomous elements rely on other transposons or host enzymes for their mobilization.

Encyclopedia of DNA Elements (ENCODE)

• Transposable elements are found in various genomes, including bacteria. Their presence and activity can vary widely across different species and even among individuals.

• ENCODE project is dedicated to identifying these elements. This project aims to comprehensively map the functional elements in the human genome, including transposable elements.

• Understanding transposable elements is crucial for:

  • Medicine (disease relevance)

  • Science (driving gene expression)

• The list helps us understand the origin and behavior of these DNA pieces like transposable elements are like viruses such as COVID-19. This comparison highlights the potential for transposable elements to act as mutagens and contribute to disease.

• Knowing more about their genetic composition help us understand how to better address how it affects us and our biology. Further research into the mechanisms of transposition and the function of transposon-derived sequences is essential.

ENCODE Project Findings

• Evaluated RNA, cytosine methylation, chromatin, and genome organization in various cell types. This multi-faceted approach provides a comprehensive view of the functional landscape of the genome.

• Approximately 80% of the genome shows biochemical evidence of function, including transposon regions. This finding challenges the notion of "junk DNA" and suggests that much of the non-coding genome plays a role in gene regulation and other cellular processes.

• Genes encoded within transposons are expressed, but their exact function is still being investigated (copying, etc.). These transposon-derived genes may contribute to novel functions or influence the expression of nearby genes.

Transposable Elements Characteristics

• Also known as mobile genetic elements.

• DNA-based, found within the genome.

• Mobile: can be removed and placed elsewhere in the genome or copied to a new location.

• Transposable: capable of moving locations.

• They no longer have the capacity to leave the cell in their own capsid like vestigial viruses. This loss of autonomy distinguishes them from active viruses.

• Both eukaryotic and prokaryotic genomes contain mobile genetic elements. Their prevalence varies across different species and can have significant impacts on genome evolution and function.

• Archaea also likely contain, but not all. Further research is needed to fully characterize the diversity and function of transposable elements in archaeal genomes.

Mechanism of Transposition

• Transposition: the process by which mobile genetic elements (transposons) move.

• Transpose: the verb of transposition.

• Transposase: an enzyme that cuts the DNA sequence containing the transposon and induces transposition. This enzyme is crucial for the mobilization of DNA-only transposons.

• Transposase recognizes specific sites on the transposable element for removal. These sites typically consist of short inverted repeats that flank the transposon.

• However, transposase does not require homology when integrating the element into a different part of the genome. This lack of homology requirement allows transposons to insert themselves into virtually any location in the genome.

Classes of Transposons: Key Features

• Recognition of transposons depends on:

  • Structure (DNA sequence)

  • Mechanism of transposition

• The three main types again:

  • DNA-only transposons

  • Retroviral-like retrotransposons

  • Non-retroviral retrotransposons

Structure of Transposable Elements

• DNA Transposons

  • Short inverted repeats.

  • Enzyme: Transposase.

  • Mechanism:

    • Two transposase enzymes cut the transposon from the host genome.

    • Then paste the transposon in a different region.

  • Found in plants (e.g., Ac/Ds in maize), Drosophila, and E. coli. These organisms have been extensively used to study the mechanisms and effects of DNA transposons.

  • P element in Drosophila is a DNA transposon used for gene integration. This element has been adapted as a tool for genetic engineering in Drosophila.

• Retroviral-like Retrotransposons

  • Long terminal repeats (LTRs) act as strong enhancers. These LTRs can drive high levels of gene expression in nearby genes.

  • Encode reverse transcriptase and integrase.

  • Reverse transcriptase may have endonuclease activity.

  • Move through an RNA intermediate.

    • Transcribed as RNA ilda mRNA ilda Proteins (reverse transcriptase and integrase)

    • Reverse transcriptase converts mRNA to cDNA ilda integrated into genome.

• Non-Retroviral Retrotransposons

  • Lack genes to encode reverse transcriptase, endonuclease, or integrase. These elements rely on other transposons or host enzymes for their mobilization.

  • Require enzymes from other transposable elements or viruses to be copied and integrated.

Mechanism of DNA-Only Transposons and Antibiotic Resistance

• Predominate in bacteria and are responsible for the spread of antibiotic resistance. The ability of these elements to move between bacteria facilitates the rapid dissemination of resistance genes.

• Transposable elements encode enzymes that inactivate antibiotics. These enzymes can modify or degrade antibiotics, rendering them ineffective.

• Mechanism: Horizontal gene transfer (copying and transferring a chromosome portion to another bacterium). This process allows bacteria to acquire new genes, including antibiotic resistance genes, from other bacteria.

• DNA-only transposons exist only as DNA.

Examples of DNA-Only Transposons

• IS3: A DNA-only transposable element with inverted repeats.

• A transposable element with transposase and ampicillin resistance gene. This element confers resistance to ampicillin, a commonly used antibiotic.

• Tn10: contains a tetracycline resistance gene and transposase gene, composed of two transposable elements. This transposon confers resistance to tetracycline and can move between bacteria, spreading resistance.

Movement Mechanisms of DNA-Only Transposons

• Two mechanisms:

  • Cut and paste transposition

  • Replicative transposition (horizontal gene transfer or DNA synthesis/proliferation)

• Cut and Paste Transposition

  • Transposase recognizes inverted repeats, loops DNA so inverted repeats align.

  • Enzyme cuts at the end of the inverted repeat.

The Cre-loxP System

• Adapted from a Drosophila flip-fret system. This system allows for precise control of gene expression and has been widely used in research.

• Sequences and enzyme come from a virus.

• Integration occurs through homologous recombination.

Details of Cut and Paste Transposition

• Transposase catalyzes a direct attack on the target DNA molecule.

• Transposase breaks two phosphodiester bonds in the target molecule (double-stranded break).

• Transposase creates new phosphodiester bonds to join the element and target DNA.

• The three prime hydroxyl group is nucleophilic and is important to conducing a nucleophilic attack to enter a new region. It is actually conducted by the transposable element itself.

• Target DNA is broken in a staggered manner leading to two short single-stranded gaps that result.

• DNA polymerase and ligase ensure the phosphate backbone seals due to that nucleophilic attack. The result is a short duplication of target DNA at the insertion site due to a staggered region that happens when the new integration occurs.

Visual Example:

1. Original DNA Sequence: ATTA on on strand and TAAT on the complementary strand of DNA.

2. Transposase Cuts:

   • The enzyme cuts, creating staggered regions.

3. Transposable Element Integration:

   • A transposable element (e.g., GC - CG) is inserted into the staggered cut.

4. Duplication and Mutation:

   • The staggered region is copied, resulting in a duplication of the sequence ATTA and TAAT.

   • This introduces additional DNA that was not originally part of the sequence, leading to a mutation.

• Repair of the original chromosome from which the transposable element was removed occurs either through non-homologous end joining or homologous recombination. These repair mechanisms ensure the integrity of the genome after transposon excision.

Retroviral-Like Retrotransposons (Mechanism #2)

• LTRs (long terminal repeats) act as enhancers, driving gene expression. These LTRs can influence the expression of genes located near the insertion site of the retrotransposon.

• Formerly retroviruses, but no longer form a capsid to leave the cell. These elements are remnants of ancient retroviral infections that have lost the ability to produce infectious particles.

• Found in yeast, flies, worms, and mammals. Their presence in diverse organisms highlights their evolutionary significance.

• No ability to leave the cell; passed along via DNA replication and cell division. This vertical transmission ensures that retrotransposons are inherited by subsequent generations.

Retroviral-Like Retrotransposons (Encoding)

• Encode:

  • Reverse transcriptase

  • Integrase

• Reverse transcriptase copies RNA as cDNA, then removes the RNA.

• Integrase then integrates cDNA into the chromosome.

Mechanism:

1. Transcription of mRNA with exons (with non-translatable elements such as five prime untranslated regions and three prime untranslated regions) that may not encode a protein.

2. Utilize the cell host's enzymes to cut, giving it a five prime cap and poly A tail.

3. It's translated to reverse transcriptase and integrase.

4. Reverse transcriptase converts RNA to cDNA, then removes the RNA via RNase H

5. Linear double-stranded DNA molecule (the cDNA) is integrated into the chromosome using the integrase enzyme.

• The three prime hydroxyl is important to catalyze the invasion reaction.

• With DNA polymerase, short direct repeats of target DNA sequence are integrated due an a nucleophilic attack.

• mRNA is polycystronic (encodes for more than one product, like the products encoded from viral infections).

Non-Retroviral Retrotransposons (Mechanism #3)

• Do not encode reverse transcriptase or endonuclease/integrase, so they hijack them to copy and then integrate into the genome. These elements rely on the enzymatic machinery of other transposons or viruses for their mobilization.

• They have a poly-T Region at the three prime end.

• Five prime end is often truncated.

• They are the most independently immobile. Their lack of coding capacity makes them dependent on other elements for their movement.

• L1 (LINE-1) inserted into blood clotting factor VIII gene, causing hemophilia. This example illustrates the potential for non-retroviral retrotransposons to cause disease.

• They utilize endonuclease and reverse transcriptase to transpose.

• Have poly a or poly T tail

• Line one or L one (long dispersed nuclear element) contains two open reading frames (encodes for two genes)

• With short interspersed nuclear element (SIN) aka ALU, which doesn't have open reading frames encodes, it needs to pirate the line open reading frames order to transpose into other enzymes.

Transposition and Endonuclease

• Mechanism:

  • Uses endonuclease and reverse transcriptase to transpose.

  • An example being in the death of the czar's last heir, possible related to World War One. This highlights the historical and medical significance of understanding transposition.

• The reverse transcriptase is going to link the RNA Initially through with the DNA through- When it has done so, it copies RNA to DNA.

  • Which then finalizes integration into that target chromosome.

Conclusion

• There are three classes of transposable element, this has described what their mechanisms are.

• Homology is not necessary for transposition.

• Transposons make up a significant fraction of the human genome, the purpose of the majority are still trying to