Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase

Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function. Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication. DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication. However, the template-directed copying of DNA is identical in all cases. The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA. The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E. coli. We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor. The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases.     

Structure of the Escherichia coli ribosomal termination complex with release factor 2

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.     

重组大肠杆菌发酵生产二倍体质粒的研究

随着基因治疗和基因疫苗的发展,急需大量的非病毒载体质粒DNA.主要对重组大肠杆菌E.coli DH5α发酵生产pUC21二倍体质粒的培养基组分和补料分批培养的葡萄糖流加策略进行了研究.初步确定的培养基组分是以葡萄糖作碳源,酵母粉作氮源,并且添加磷酸盐、硫酸镁、柠檬酸和微量元素.研究发现溶氧反馈流加是比较好的流加葡萄糖的补料策略,它能把葡萄糖浓度控制在较低的水平,从而避免产生乙酸效应.溶氧反馈流加发酵的最大生物量可达30.84 g/L,质粒pUC21-Dimer的最大产量达96.38 mg/L.该研究为重组大肠杆菌生产二聚体质粒建立了优化工艺,对大规模生产作为基因治疗的多聚体质粒具有指导意义.     

Inducible repair of oxidative DNA damage in Escherichia coli

Hydrogen peroxide is lethal to many cell types, including the bacterium Escherichia coli. Peroxides yield transient radical species that can damage DNA and cause mutations. Such partially reduced oxygen species are occasionally released during cellular respiration and are generated by lethal and mutagenic ionizing radiation. Because cells live in an environment where the threat of oxidative DNA damage is continual, cellular mechanisms may have evolved to avoid and repair this damage. Enzymes are known which evidently perform these functions. We report here that resistance to hydrogen peroxide toxicity can be induced in E. coli, that this novel induction is specific and occurs, in part, at the level of DNA repair.     

AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli

The bacterial AlkB protein is known to be involved in cellular recovery from alkylation damage; however, the function of this protein remains unknown. AlkB homologues have been identified in several organisms, including humans, and a recent sequence alignment study has suggested that these proteins may belong to a superfamily of 2-oxoglutarate-dependent and iron-dependent oxygenases (2OG-Fe(ii)-oxygenases). Here we show that AlkB from Escherichia coli is indeed a 2-oxoglutarate-dependent and iron-dependent DNA repair enzyme that releases replication blocks in alkylated DNA by a mechanism involving oxidative demethylation of 1-methyladenine residues. This mechanism represents a new pathway for DNA repair and the third type of DNA damage reversal mechanism so far discovered.