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Related Concept Videos

Overview of Transposition and Recombination02:13

Overview of Transposition and Recombination

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Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
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Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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Protein Editing using a Concerted Transposition Reaction.

Yi Hua1, Nicholas E S Tay1, Xuanjia Ye1

  • 1Department of Chemistry, Princeton University, Princeton, NJ, USA.

Biorxiv : the Preprint Server for Biology
|June 19, 2024
PubMed
Summary

This study introduces protein transposition, a novel method for swapping protein segments in a single step. This technique enables efficient protein engineering under native folding conditions, expanding possibilities for biochemical research.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Protein ligation is powerful for in vitro biochemical studies.
  • Current methods require protein folding after fragment ligation, limiting applications.

Purpose of the Study:

  • To develop a novel in vitro strategy for replacing internal protein regions in a single operation.
  • To enable protein manipulation within a native fold.

Main Methods:

  • Developed a 'protein transposition' system using orthogonal split inteins.
  • Analogous to DNA transposition, it swaps protein segments.
  • Utilized isotopic labeling to confirm reaction kinetics.

Main Results:

  • Demonstrated a concerted protein transposition reaction when intein pair kinetics are matched.
  • Successfully applied the method to various protein systems, including the ACF complex.
  • Showed that protein transposition can occur in situ within the cell nucleus.

Conclusions:

  • Protein transposition allows efficient manipulation of protein primary structure under native folding conditions.
  • This method significantly expands the scope of protein semisynthesis.
  • The approach is applicable to complex protein systems and in vivo applications.