DNA gyrase is unique among enzymes for its ability to actively introduce negative supercoils into DNA. This function is mediated in part by the C-terminal domain of its A subunit (GyrA CTD). Here, we report the crystal structure of this Ϸ35-kDa domain determined to 1.75-Å resolution. The GyrA CTD unexpectedly adopts an unusual fold, which we term a -pinwheel, that is globally reminiscent of a -propeller but is built of blades with a previously unobserved topology. A large, conserved basic patch on the outer edge of this domain suggests a likely site for binding and bending DNA; fluorescence resonance energy transfer-based assays show that the GyrA CTD is capable of bending DNA by >180°over a 40-bp region. Surprisingly, we find that the CTD of the topoisomerase IV A subunit, which shares limited sequence homology with the GyrA CTD, also bends DNA. Together, these data provide a physical explanation for the ability of DNA gyrase to constrain a positive superhelical DNA wrap, and also suggest that the particular substrate preferences of topoisomerase IV might be dictated in part by the function of this domain.T he topology of cellular DNA is managed by topoisomerases, enzymes that pass DNA strands through each other to relieve excess supercoiling and resolve DNA knots and catenanes (1, 2). Whereas all organisms contain at least one topoisomerase, the bacterium Escherichia coli possesses four, each with distinct roles: topoisomerase (topo) I, topo III, topo IV, and DNA gyrase. Topo I and topo III pass single DNA strands through one another to relax negative supercoils (1) or aid RecQ-family helicases in certain DNA repair processes, respectively (3). DNA gyrase and topo IV use ATP to power the transport of one intact DNA duplex through another, an activity that can alter DNA superhelicity as well as promote chromosome decatenation (4, 5).DNA gyrase and topo IV are members of the type IIA topoisomerase superfamily (6, 7). These enzymes are assembled as oligomeric complexes with distinct domains that coordinate ATP binding and hydrolysis with DNA binding, cleavage, and transport (7,8). The type IIA topo reaction cycle begins when one segment of DNA, termed the G segment, binds across the central region of the enzyme. ATP binding then triggers a series of motions that leads to the capture of a second DNA duplex (the T segment), cleavage and opening of the G segment, and passage of the T segment through the break. Once the T segment is transported, the G segment is resealed, the T segment is expelled from the protein, and ATP is hydrolyzed and released. This enzymatic cycle alters the linking number of the substrate DNA in discrete steps of Ϯ 2.Although all type IIA topos share this basic mechanism, there exist distinct type IIA subtypes that have differing substrate specificities and activities. The eukaryotic enzyme, topo II, relaxes positively and negatively supercoiled DNAs at the same rate (9) and can decatenate chromosomes (5). In contrast, most bacteria possess two somewhat more specialized type IIA topos; DNA ...
Information theory was used to build a promoter model that accounts for the −10, the −35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base −7, rather than −11, of the −10 may be flipping to initiate transcription. We found that the sequence conservation of σ70 binding sites is 6.5 ± 0.1 bits. Some promoters lack a −35 region, but have a 6.7 ± 0.2 bit extended −10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended −10 binding and the −35 suggest that the flexible bipartite σ factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5′ mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the −35, so bending allows distal activators to reach the polymerase.
A pulse-chase approach is outlined for measuring mRNA turnover rates under changing growth conditions.
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