分子生物学,基因工程,DNA结构、复制与转座培训资料.pptVIP

the clamp-loading complex The clamp-loading complex consists of the delta (δ) (38.7 kD) and delta (δ) prime (36.9 kD), chi (χ) (16.6 kD) and psi (ψ) (15.2 kD) subunits and either or both of the gamma (γ) subunit (68.4 kD) and the tau (τ) subunit (71.1 kD). Both the gamma and tau subunits are encoded by the dnaX gene. This gene is translated in two different ways. If the gene is translated completely then the tau (τ) subunit is synthesized. If translation slips or frameshifts part way through the reading frame then a stop codon is encountered, and the gamma (γ) subunit is synthesized.Both the gamma (γ) subunit and the tau(τ) subunit are ‘motor ATPases. The structures of many of these subunits have now been solved and they are beginning to reveal how the different subunits interact and function in the building of a replisome and in its ongoing function. The δsubunit binds to theβ subunit and, in concert with theδ andγsubunits and with ATP, it catalyses the opening of theβ dimer to permit passage of DNA.  The catalytic mechanism likely involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases. The top Mg2+ ion in the figure facilitates attack of the 3’-hydroxyl group of the primer on the phosphate of the nucleotide triphosphate; the lower Mg2+ ion facilitates displacement of the pyrophosphate. Both ions stabilize the structure of the pentacovalent transition state. RNA polymerases use a similar mechanism . (4) Termination Locations and sequences of the replication termini of E. coli. (A) Map showing the ori and the 10 Ter sites. (B) The consensus sequence of Ter. FIGURE Termination of chromosome replication in E. coli. (a) The Ter sequences are positioned on the chromosome in two clusters with opposite orientations. (b) Replication of the DNA separating the opposing replication forks leaves the completed