PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-Å resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique ␤-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.DNA polymerase III ͉ DNA replication ͉ Gram-positive polymerase ͉ polymerase and histidinol phosphatase (PHP) ͉ ternary complex D NA polymerases are the enzymes responsible for DNA synthesis. Cellular organisms typically use multiple DNA polymerase types. The ''replicative'' polymerase performs the bulk of genome duplication, whereas various specialty polymerases repair damaged DNA and resolve Okazaki fragments. Across every kingdom of life, replicative polymerases exhibit certain hallmarks such as high fidelity, speed, and processivity (1). Polymerase holoenzyme accessory proteins play an integral role in achieving the extraordinary efficiency and accuracy of the replicative polymerase complex. These include a ''sliding clamp'' that encircles the DNA and increases processivity (2).Bacterial replicative polymerases comprise the C family of DNA polymerases (3) and differ significantly from the replicative polymerases of eukaryotes, bacteriophage, and archaea, which belong to the B family. The major C family replicative polymerases are DnaE (PolIII), found primarily in Gram-negative bacteria, and PolC (PolIIIC), found primarily in Gram-positive bacteria (4). Apoenzyme crystal structures of DnaE have revealed surprising structural differences in the catalytic center of the enzyme compared with B family polymerases, suggesting a separate evolutionary origin for the C family (5, 6).As the core component of the replicative polymerase complex in Gram-positive pathogens such as Staphylococcus aureus, PolC has received considerable attention as a potential target for antibacterial drug discovery (7,8). No currently marketed antibiotics target the central replication apparatus, making PolC a novel target for antibacterial development. Gram-negative and Gram-positive bacteria are separated by Ͼ1 billion years of evolution, and PolC and DnaE share Ͻ20% sequence identity; PolC is further differentiated from DnaE by domain rearrangements and by the presence of an ...

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REP3123 demonstrated a favourable microbiological profile and was found to target C. difficile with high specificity and selectivity.

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu⅐GTP⅐aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu⅐GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu⅐Ts). Mammalian mitochondrial EF-Tu (EF-Tu mt ) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu mt subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K tRNA ) and for the dissociation of the EF-Tu⅐Ts mt complex (K Ts ) were investigated. The equilibrium dissociation constant for the ternary complex was 18 ؎ 4 nM, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 ؋ 10 ؊4 s ؊1 , which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu mt to EF-Ts mt is mutually exclusive with the formation of the ternary complex. K Ts was determined by quantifying the effects of increasing concentrations of EF-Ts mt on the amount of ternary complex formed with EF-Tu mt . The value obtained for K Ts (5.5 ؎ 1.3 nM) is comparable to the value of K tRNA .The classical model of the elongation cycle for protein synthesis is based on studies with Escherichia coli. In this model, the active form of elongation factor (EF) 1 Tu (the EF-Tu⅐GTP complex) binds aa-tRNA, forming a ternary complex. This ternary complex promotes the binding of aa-tRNA to the A-site of the ribosome. Once locked into the A-site by cognate codonanticodon interactions, the GTP in the ternary complex is hydrolyzed, and EF-Tu is released from the ribosome as an EFTu⅐GDP complex. This complex must be dissociated by EF-Ts through the formation of an intermediate EF-Tu⅐Ts complex. GTP then replaces EF-Ts, reforming the active form of EF-Tu.The equilibrium dissociation constant governing the affinity of E. coli EF-Tu for GDP is 8 nM, compared with 0.3 M for GTP (1). The dissociation of the EF-Tu⅐GDP complex is thought to be the rate-limiting step in the recycling of EF-Tu (1). EF-Ts promotes the exchange of the tightly bound GDP for GTP through the formation of an intermediate EF-Tu⅐Ts complex. Presumably one of the reasons for the requirement for EF-Ts is because of the 40-fold higher affinity of EF-Tu for GDP compared with GTP. E. coli EF-Tu and EF-Ts have a high affinity for each other; the dissociation constant for the E. coli EFTu⅐Ts complex (K Ts ) is 2 nM (2). However, under intracellular conditions in E. coli, the EF-Tu⅐Ts complex appears to have a transient existence. The EF-Tu⅐Ts complex is rapidly converted to the ternary complex by high intracellular concentrations of GTP and by the tight binding of aa-tRNA to the EF-Tu⅐GTP complex. The dissociation constant for the release of aa-tRNA from the ternary complex is ϳ1 nM in E. coli (3). EF-Tu is a highly conserve...

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