Search

The replication system of bacteriophage T4 uses a trimeric ring-shaped processivity clamp (gp45) to tether the replication polymerase (gp43) to the template-primer DNA. This ring is placed onto the DNA by an ATPase-driven clamp-loading complex (gp44/62) where it then transfers, in closed form, to the polymerase. It generally has been assumed that one of the functions of the loading machinery is to open the clamp to place it around the DNA. However, the mechanism by which this occurs has not been fully defined. In this study we design and characterize a double-mutant gp45 protein that contains pairs of cysteine residues located at each monomer-monomer interface of the trimeric clamp. This mutant protein is functionally equivalent to wild-type gp45. However, when all three monomer-monomer interfaces are tethered by covalent crosslinks formed (reversibly or irreversibly) between the cysteine pairs these closed clamps can no longer be loaded onto the DNA nor onto the polymerase, effectively eliminating processive strand-displacement DNA synthesis. Analysis of the individual steps of the clamp-loading process shows that the ATPase-dependent interactions between the clamp and the clamp loader that precede DNA binding are hyperstimulated by the covalently crosslinked ring, suggesting that binding of the closed ring induces a futile, ATP-driven, ring-opening cycle. These findings and others permit further characterization and ordering of the steps involved in the T4 clamp-loading process.

The kinetics of the ATP-dependent RNA-DNA helicase activity of Escherichia colitranscription termination factor rho have been analyzed. Helicase substrates were assembled using 255 nt and 391 nt RNA sequences from the trp t' RNA transcript of E. coli. These RNA sequences each carry a rho "loading site" at a position near the 5'-end, and a rho-dependent terminator sequence at the 3'-end to which complementary approximately 20 nt DNA oligonucleotides have been annealed. A rapid ( approximately 30 s) pre-steady-state burst of helicase activity (DNA oligomer release), followed by a slow linear phase, is observed in reactions carried out at low salt concentrations (50 mM KCl). Using poly(rC) or poly(dC) as traps for the rho that is released after one round of activity, we have shown that the first (burst) phase of the reaction represents the processive translocation of prebound rho hexamers from the rho loading site to the 3'-end of the RNA molecule. The slow phase of the reaction is complex and represents a combination of many different processes, including the slow release of RNA from rho, the reannealing of complementary DNA oligonucleotides to the RNA substrate, and the recycling of rho hexamers onto additional RNA molecules. Reactions carried out at higher salt concentrations (150 mM KCl) consist of only one phase, since under these conditions rho dissociates more rapidly from the RNA, with an amplitude corresponding to several DNA oligomers removed per rho hexamer. Thus, rho can recycle and function as a catalytic helicase under reaction conditions resembling those found in the cell.

The ATPase and helicase activities of the Escherichia coli transcription termination protein rho have been studied under a variety of reaction conditions that alter its transcription termination activity. These conditions include KCl, KOAc, or KGlu concentrations from 50 to 150 mM and Mg(OAc)2 concentrations from 1 to 5 mM (in the presence of 1 mM ATP). In higher KCl or higher Mg(OAc)2 concentrations we found that the translocation of rho hexamers along RNA was slower and less processive than the same process measured at 50 mM monovalent salt concentrations and 1 mM Mg(OAc)2. The ATPase activity of rho was also decreased under reaction conditions that slowed translocation. RNA melting experiments showed that the decreased ATPase activity of rho and the slower helicase activity at increased KCl or Mg(OAc)2 concentrations are accompanied by a concomitant increase in the secondary structure of the RNA portion of the helicase substate. In contrast, the ATPase activity of rho in the presence of poly(rC), a synthetic RNA that does not form salt-concentration-dependent secondary structure, was shown to be the same in each of the three monovalent salts. Thus, the salts do not directly affect the structure or conformation of the rho protein or the binding of rho to single-stranded RNA. However, the translocation of rho along RNA was more processive in 150 mM KOAc or KGlu than in 150 mM KCl, while the RNA secondary structure was the same in all three monovalent salts. Therefore, the monovalent salt present in the reaction may directly affect rho-RNA interactions when the RNA substrate can form secondary structure. Helicase experiments with an RNA molecule that does not contain a rho loading-site showed that rho translocates less processively along this potential helicase substrate. These results suggest that the helicase activity of rho may be significantly regulated by RNA secondary structure. In addition, one of the mechanisms to concentrate the activity of rho on transcripts containing unstructured rho loading sites may be that rho translocation along such molecules is more processive than it is along more structured RNA molecules in the cell.

The RNA-binding and RNA-DNA helicase activities of the Escherichia coli transcription termination factor rho have been investigated using natural RNA molecules that are 255 and 391 nucleotide residues in length and that contain the trp t' rho-dependent termination sequence of E. coli. Helicase substrates were prepared from these RNA molecules by annealing one or more DNA oligomers to complementary sequences located at or near the 3'-ends of the RNA molecules to form defined RNA-DNA hybrid sequences ranging in length from 20 to 100 bp. By comparing the fraction of the RNA molecules bound to rho with the fraction of bound DNA oligomers removed from the RNA during one round of the helicase reaction, we have shown that rho translocates processively at 37 degrees C in buffer containing 50 mM KCl. Helicase reactions and ATPase measurements were performed in parallel in the presence of RNA molecules containing RNA-DNA hybrids of various lengths, and we show that both the rate of translocation of the rho hexamer along the RNA chain and the rate of ATP consumption are similar, whether or not DNA is hybridized to the RNA transcript. By combining measurements of translocation and ATPase rates, we estimate that rho consumes approximately 1-2 ATP molecules in translocating over 1 nucleotide residue of the RNA chain at 37 degrees C in 50 mM KCl. The ATPase activity of rho remains the same after one round of the helicase reaction, indicating that rho appears to hydrolyze ATP at the same rate, whether it is translocating along the RNA, separating RNA-DNA hybrids, or bound at the 3'-end of the RNA substrate. We also show that rho binds cooperatively ( approximately 2-4 rho hexamers per RNA chain) to the RNA substrates under our standard helicase reaction conditions. However, cooperative binding is not essential for helicase activity, since this binding stoichiometry can be reduced to approximately 1.5 rho hexamers per 255-nucleotide residue RNA chain by blocking approximately 100 nt of either end of the rho binding site of the helicase substrate with complementary DNA oligonucleotides, with no change in helicase properties. The implications of these results for models of rho helicase function and for the role of rho in termination are discussed.