Transposons Tn10 and Tn5

Structure of Tn10 and Tn5. (A) Tn10 is a composite transposon that encodes resistance determinants for tetracycline. The OEs and IEs of IS10-Right and IS10-Left are shown as well as the transcription units of IS10-Right. RNA-IN encodes the transposase and RNA-OUT encodes an antisense RNA. (B) Pairing of RNA-IN and RNA-OUT. RNA-OUT is a highly structured antisense RNA to the transposase RNA, RNA-IN. There are 35 nucleotides of perfect base complementarity between RNA-IN and RNA-OUT. Pairing initiates between the 5′ end of RNA-IN and the hairpin loop of RNA-OUT and full pairing results in the sequestration of both the Shine–Dalgarno (SD) sequence of RNA-IN and the start codon (AUG). Positions of the two main internal bulges in the stem are indicated. (C) Structure of Tn5. Tn5 is a composite transposon that encodes resistance to kanamycin, bleomycin, and streptomycin. The OE and IE of IS50-Right and IS50-Left are shown as well as the transcription units in IS50-Right. Transcripts Tnp and Inh encode the transposase and inhibitor proteins, respectively.

Tsome dynamics in Tn10 transposition. The top line shows the initial Tsome with an IHF-folded arm (α-arm) transitioning to a single end break complex (αSEB) in which flanking donor DNA has been cleaved from one end and the α-arm remains folded, to an unfolded single end break complex (uf-SEB), and then to an unfolded double end break complex (uf-DEB) where flanking donor DNA has been cleaved from both transposon ends. At the branch point the uf-DEB can either capture a target DNA (TCC, target-capture complex) that is not part of the transposon and catalyze an intermolecular strand-transfer event (STC, strand-transfer complex) or the uf-DEB can rebind IHF to refold a transposon arm and undergo an intramolecular target-capture and strand-transfer event in which part of the transposon serves as the target DNA. The lower line shows the impact of H-NS on Tsome dynamics. An H-NS dimer is shown binding to the flanking DNA of the β-arm of the SEB that contains a distorted DNA structure (squiggly line in top panel). H-NS then facilitates the displacement of IHF (through an unknown mechanism) permitting the α-arm to unfold and subsequently additional H-NS dimers are recruited to the unfolded Tsome. H-NS binding within the transposon sequences is proposed to help maintain the Tsome in an unfolded form to both stabilize the fully cleaved unfolded Tsome and promote intermolecular target capture. Note that H-NS might first interact with the initial Tsome (denoted bPEC for historical reasons) instead of the α-SEB as shown. Transposon end sequences, arrows attached to black lines; flanking donor DNA, grey lines; target DNA, dashed lines; transposase, ovals. For clarity the two transposon ends are not joined (indicated by double dashes).

Strand-transfer products formed in Tn10 transposition. If IHF (orange circle) maintains at least one OE in the folded form after excision (lower branch of diagram), insertion events are “channeled” into a target site (red line) located close to an OE within the transposon. Accordingly, intramolecular STPs are formed, and these products are “topologically simple” (UKIC) and unlinked deletion circles (DCs) because supercoiling nodes present in the excision product are not trapped between the OEs and the target site (dotted red line shows the recombination event; intrastrand strand transfer leads to the formation of DCs, whereas interstrand strand transfer leads to formation of ICs). If IHF does not remain bound to the OE and the OE unfolds, then the OE can interact with a target site through “random collision” (upper branches of diagram). If the target site is on a separate DNA molecule, intermolecular transposition occurs, whereas if the target site is within the transposon, intramolecular transposition occurs. The products of the latter include topologically complex (T-Cpx) DCs (catenated) and ICs (knotted). Green lines indicate transposon DNA; black lines, flanking donor DNA; ovals, transposase. Reprinted from the Journal of Molecular Biology ( 39 ) with permission from Elsevier.

Hfq-binding sites within RNA-IN and RNA-OUT. A portion of IS10-Right is depicted along with partial sequences of the first 95 and 40 nucleotides of RNA-IN and RNA-OUT, respectively. The positions of Hfq-binding sites defined by in vitro footprinting are also shown (thick lines). Blue and red lettering distinguish sites thought to interact with the proximal and distal binding faces of Hfq, respectively. The SD and start codon of RNA-IN are underlined (thin lines). Note that the distal Hfq-binding site at the 5′ end of RNA-IN overlaps with the SD sequence.

Structure and RNA-binding properties of Hfq. Hfq monomers assemble into a homohexameric ring-shaped complex with three distinct RNA-binding surfaces. The proximal surface (located close to the N-terminus) preferentially binds short U-rich stretches of RNA with each nucleotide binding to a pocket formed from adjacent protomers. The opposite face of the hexamer is termed the distal surface and binds longer RNA sequences that are purine rich. Each monomer contains three nucleotide-binding pockets termed the A, R, and N sites, which interact with adenines, purines, and the sugar-phosphate backbone, respectively. Each hexamer also possesses six additional RNA binding sites (lateral sites, not shown) positioned within each monomer between the distal and proximal sites. A lateral site can accommodate RNA extending from the proximal and distal sites. Hfq monomers are in red, blue, and green and RNA is in gold. Adapted from Nature Reviews: Microbiology ( 27 ) with permission from Macmillan Publishers.