Summary
Highlights
The lecture begins by reviewing conservative site-specific recombination, emphasizing that in such processes, the number of broken bonds equals the number of formed bonds, specifically phosphodiester bonds. It revisits the two main types of recombinases: serine and tyrosine recombinases. Serine recombinases perform double-strand breaks and exchange entire DNA segments, while tyrosine recombinases make single-strand breaks, forming a Holliday junction before proceeding to a second break and exchange.
An interesting biological example is presented with Salmonella's ability to switch between H1 and H2 flagellin types. This is achieved through the inversion of a DNA segment containing a promoter, regulated by the HixR and HixL sites and the Hri recombinase. The orientation of this invertible segment determines which flagellin gene (H1 or H2) is expressed, illustrating how site-specific recombination influences phenotypic expression and bacterial survival.
The discussion transitions to transposons, genetic elements capable of moving within the genome. The instructor highlights the philosophical and historical significance of Barbara McClintock's Nobel Prize-winning work on transposons in maize, initially met with skepticism. Transposons are considered genetic information despite not always coding for proteins, as they are associated with replicon and can replicate independently through transposition.
Transposons are classified into three main types: DNA transposons, virus-like retrotransposons, and retrotransposons with poly-A tails. The basic architecture of DNA transposons is described, including the requirement for terminal inverted repeats and a transposase enzyme. The concepts of autonomous (self-sufficient) and non-autonomous (dependent on other transposons) elements are explained, along with 'grounded' transposons that are unable to move due to mutations in their essential elements.
The 'cut and paste' mechanism of DNA transposons is detailed. The transposase enzyme recognizes inverted terminal repeats, excises the transposon, and inserts it into a new target site. This process results in target site duplications (TSDs) flanking the inserted transposon, which serve as a signature of past transposition events. The lecture illustrates how these TSDs are formed and can be identified.
Different mechanisms for the second strand break during excision are presented, using examples like Tn7, Tn10/Tn5, and Hermes transposons. These variations demonstrate that while the basic cut-and-paste principle remains, the molecular details of how the transposon is fully excised can differ, involving specialized proteins, hairpin formation, or nucleophilic attacks by the host DNA.
The 'copy and paste' mechanism, also known as replicative transposition, is explained. In this process, the transposon attacks a target site, forms an intermediate cointegrate structure, and then new DNA is synthesized at replication forks created within this intermediate. This results in the original transposon remaining in its donor site, while a new copy is integrated into the target site, effectively increasing the number of transposons in the genome.
The structure and basic mechanism of retroviral-like transposons and poly-A retrotransposons are introduced. Retroviral-like transposons have long terminal repeats (LTRs), integrase, and reverse transcriptase genes. They are transcribed into RNA, which is then reverse-transcribed into DNA and integrated into the genome by integrase. Poly-A retrotransposons feature open reading frames (ORF1 and ORF2), untranslated regions (UTRs), and a characteristic poly-A tail, distinguishing them from other types of retrotransposons.
The lecture explains how retrotransposons, specifically the LTR type, utilize transcription to produce RNA, which is then translated into integrase and reverse transcriptase. The reverse transcriptase synthesizes a double-stranded DNA copy of the transposon, which is subsequently integrated into a new target site by integrase, leading to target side duplications and an increase in transposon copy number within the genome. The next class will cover poly-A retrotransposons and immunology topics.