Topoisomerase IV belongs to the type II class of DNA topoisomerases, which are responsible for changing and stabilizing DNA supercoiling and are also involved in chromosome segregation in prokaryotes. Topo IIs are essential enzymes for bacterial replication and are targeted by antibacterial drugs such as quinolones or diones. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA (G-segment) duplex and transporting the second duplex (T-segment) though a doublestranded break in the formed protein-DNA gate. Although the biological importance of these enzymes is well known, cleavage complex formation and reversal is not fully understood for any type II topoisomerase. In order to further our understanding of the topo II action, we have solved the crystal structures representing sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from S. pneumoniae. A 3.1 Å resolution structure of the complex captured by a novel antibacterial dione represents a drugarrested form of the cleavage intermediate and reveals two drug molecules intercalated at a symmetrically cleaved B-form DNA gate and stabilized by drug-specific protein contacts. Similar protein/DNA/drug complex formation was observed for the 2.9 Å resolution structure of topo IV/DNA/levofloxacin solved by us and representing the first high-resolution quinolone-stabilized cleavage complex. Subsequent dione release allowed us to obtain drug-free cleaved and resealed DNA complexes in which the DNA gate, in contrast to the previous state, adopts an unusual A/B-form helical conformation. It also revealed an important reposition of a Mg 2+ ion towards scissile phosphodiester group allowing its coordination and promoting reversible cleavage by activesite tyrosines. These are the first structures solved for putative reaction intermediates of a type II topoisomerase. They indicate how a type II enzyme reseals DNA during its normal reaction cycle as well as how the complex is stabilized by different antibacterial drugs, which is important for the development of new topoisomerase-targeting therapeutics. U does not only stabilize the fold of the tRNA, but also plays a central role in bacterial UV protection acting as a sensor for near-UV radiation. U8 is post-transcriptionally modified by a set of enzymes including the 4-thiouridine synthetase ThiI. Here we report the crystal structure of ThiI from T. maritima in complex with a truncated substrate tRNA. The structure demonstrates that ThiI functions only as homo-dimer, since the tRNA acceptor stem including the 3'-recognition element ACCA is bound by the N-terminal ferredoxin-like and THUMP domains of one monomer thereby correctly positioning U8 close to the active site in the pyrophosphatase domain of the other monomer. The structure also indicates that full-length substrate tRNA has to adopt a non-canonical conformation upon binding to ThiI. Keywords