Transposition and Site-Specific Recombination

Transposition and Site-Specific Recombination

  • Mobile Genetic Elements

    • Length: Ranges from 100's to 10,000's base pairs long

    • Movement: Capable of moving through the genome

  • Location of Mobile Genetic Elements

    • Homology: The locations for mobile genetic elements do not have to be homologous.

  • Mechanism of Movement

    • Utilization of Enzymes: Mobile genetic elements utilize enzymes to facilitate their movement and insertion into DNA.

  • Loss of Mobility

    • Possible for mobile genetic enzymes to lose their ability to move.

  • Utility of Mobile Genetic Enzymes

    • Function: They can alter areas of DNA or enhance genetic diversity.

  • Categories of Movement

    • Transposition

    • Utilizes transposons

    • Conservative Site-Specific Recombination

    • Common in prokaryotes

  • Transposase

    • Location: The sequence for transposase is found on the DNA of the transposon itself.

    • Function:

    • Cuts out the transposon from its original location and transports it to a random site in the genome, then inserts it into the new location.

  • Retroviral-like Retrotransposons

    • Description: These transposons contain genes that encode several proteins including integrase and reverse transcriptase.

  • Reverse Transcriptase (RT)

    • Function: Catalyzes the synthesis of a new double-stranded DNA molecule from its own mRNA molecule.

  • Integrase

    • Function: Catalyzes the insertion of the newly synthesized DNA into another location in the host genome.

  • Characteristics of Retroviral-like Retrotransposons

    • Similar to retroviruses in mechanism but differ because they lack protein coats, preventing them from exiting the cell, thus are not considered true retroviruses.

  • Comparison of Retroviruses and Retrotransposons

    • Retroviruses: Encode mRNA that codes for coat proteins allowing for movement between cells.

    • Retrotransposons: Do not code for coat proteins and remain within cells.

Relationship of DNA Processes

  • Evolutionary Relationship

    • DNA repair, homologous recombination, transpositional recombination, and site-specific recombination are all evolutionarily related processes.

    • Common Mechanism: Involve site recognition, cutting and/or excision of nucleotides, insertion, and replication of new sequences.

Transcription Overview

  • Definition: The process by which a short segment of DNA is copied into RNA.

  • Types of RNA Required for Transcription

    • mRNA/pre-mRNA, rRNA, tRNA

  • Bacterial Immune System

    • CRISPR: Utilizes crRNA (clustered regularly interspaced short palindromic repeats) as a form of immune system for the DNA of bacteria and archaea.

  • Definition of a Gene

    • A gene is defined as a copied portion of DNA.

  • Structure of RNA

    • Composed of: Phosphate, ribose, and a nitrogenous base (notably, uracil instead of thymine).

    • RNA is typically single-stranded and can exhibit tertiary structure.

Prokaryotic Transcription Steps

  1. Initiation

    • Process:

      • Sigma factor binds to RNA polymerase.

      • The complex slides along the DNA until a promoter sequence is encountered.

      • The sigma factor subunit binds to the promoter.

      • A conformational change in the sigma factor opens the double helix.

      • The sigma factor then dissociates.

  2. Elongation

    • Process:

      • After the sigma factor dissociates, RNA polymerase forms a jaw-like structure to hold the DNA in place.

      • Ribonucleoside triphosphate (rNTP) uptake occurs, and RNA exit channels are formed on the protein.

      • A rudder-like protrusion also forms to separate RNA from DNA.

  3. Termination

    • Process:

      • RNA polymerase reaches the terminator sequence and transcribes it, forming an RNA “hairpin.”

      • The hairpin structure forces the RNA “jaw” open, resulting in the release of RNA from DNA.

      • RNA polymerase then dissociates from the DNA.

  • Comparison of Eukaryotic RNA Polymerase

    • Eukaryotes possess three types of RNA polymerases (POL I, POL II, POL III) as opposed to a single RNA polymerase in prokaryotes.

    • Functions: While they differ in specific roles, eukaryotic RNA polymerases function similarly to one another and to bacterial RNA polymerase.

  • Eukaryotic RNA Polymerase II

    • Responsible for transcribing pre-mRNA.

Eukaryotic Initiation Process

  • General Process:

    • General transcription factors (TFs) assemble on the double helix along with RNA polymerase before binding occurs.

    • TFIID binds to the promoter sequence approximately 25 bp upstream from the transcription start site, distorting the DNA helix and creating a high-affinity area for additional TFs, forming the transcription initiation complex.

    • TFIIH binds to RNA polymerase II, functioning as a helicase, enabling RNA polymerase II to be inserted into the DNA.

    • After creating a small amount of pre-mRNA, TFIIH phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II.

    • Subsequently, RNA polymerase II leaves the promoter, general transcription factors dissociate, and elongation commences.