Majety Naga Leelaram

Topoisomerase I (TopoI) is the sole DNA relaxase in Mycobacterium tuberculosis. Despite being a validated drug target and indispensable to the pathogen, only a limited repertoire of inhibitors targeting the enzyme have been identified. We employed monoclonal antibodies (mAbs) to address this shortfall. From the pool of a large number of mAbs, we describe an inhibitory mAb specific to mycobacterial TopoI with a distinct mechanism of action. Among the various steps of the TopoI reaction cycle, the mAb does not interfere with DNA binding but impedes DNA cleavage. It does not alter the religation activity of TopoI; however, it inhibits its strand passage activity. Probing with the mAb, we show the precise step at which the topology of the DNA is changed during DNA relaxation reaction. Surprisingly, instead of the initial strand scission action of the enzyme, the subsequent strand passage followed by the second transesterification entails the alteration in DNA topology. With their selective and specific inhibitory properties, the mAb and its derived single-chain variable fragment (ScFv) would serve to probe the structure of mycobacterial TopoI and as a starting point in designing peptide inhibitors with therapeutic potential to combat the rampant drug-resistant M. tuberculosis.

Splicing machine shifts into gear: Spliceosome activation involves extensive protein exchanges and RNA rearrangements that lead to the formation of a catalytically active U2/U6 RNA structure called B ^act . Previously, little was known about the pathway leading to the U2/U6 active site and how proteins aid in folding the U2/U6 RNA. Using cryo–electron microscopy to determine structures of two human pre-B ^act complexes, Townsend et al. uncovered an intricate cascade of coordinated structural changes involving mutually exclusive interactions that facilitate the directionality of the activation process. These structures reveal the assembly pathway of the U2/U6 catalytic RNA and the mechanism whereby proteins facilitate its folding. Science , this issue p. eabc3753