RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II

Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.     

RNA polymerase pushing

Molecular motors can exhibit Brownian ratchet or power stroke mechanisms. These mechanistic categories are related to transition state position: An early transition state suggests that chemical energy is stored and then released during the step (stroke) while a late transition state suggests that the release of chemical energy rectifies thermally activated motion that has already occurred (ratchet). Cellular RNA polymerases are thought to be ratchets that can push each other forward to reduce pausing during elongation. Here, by constructing a two-dimensional energy landscape from the individual landscapes of active and backtracked enzymes, we identify a new pushing mechanism which is the result of a saddle trajectory that arises in the two-dimensional energy landscape of interacting enzymes. We show that this mechanism is more effective with an early transition state suggesting that interacting RNAPs might translocate via a power stroke.     

Constitution of the twin polymerase of DNA polymerase III holoenzyme

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.     

Directed polymerase evolution

Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.     

DNA polymerase beta

Mammalian DNA polymerase beta(beta-pol) is a single polypeptide chain enzyme of 39kDa. beta-pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of beta-pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the beta-pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of beta-pol's role in protecting genomic DNA.     

A dynamic polymerase exchange with Escherichia coli DNA polymerase IV replacing DNA polymerase III on the sliding clamp

An assay that measures synchronized, processive DNA replication by Escherichia coli DNA polymerase III holoenzyme was used to reveal replacement of pol III by the specialized lesion bypass DNA polymerase IV when the replicative polymerase is stalled. When idled replication is restarted, a rapid burst of pol III-catalyzed synthesis accompanied by approximately 7-kb full-length products is strongly inhibited by the presence of pol IV. The production of slower-forming, shorter length DNA reflects a rapid takeover of DNA synthesis by pol IV. Here we demonstrate that pol IV rapidly (<15 s) obstructs the stable interaction between pol III* and the beta clamp (the lifetime of the complex is >5 min), causing the removal of pol III* from template DNA. We propose that the rapid replacement of pol III* on the beta clamp with pol IV is mediated by two processes, an interaction between pol IV and the beta clamp and a separate interaction between pol IV and pol III*. This newly discovered property of pol IV facilitates a dynamic exchange between the two free polymerases at the primer terminus. Our study suggests a model in which the interaction between pol III* and the beta clamp is mediated by pol IV to ensure that DNA replication proceeds with minimal interruption.     

Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase

Polymerase chain reaction (PCR) was performed with two polymerases. Thermus aquaticus DNA polymerase (Taq), and modified T7 DNA polymerase (Sequenase). Both polymerases were used to amplify the same portion of the human 18S rRNA gene. We report a PCR artifact, namely a deletion of 54 bp, when Taq polymerase was used to amplify a portion of the human 18S rRNA gene. PCR performed with Sequenase did not produce this artifact. The deletion eliminated a potentially stable hairpin loop. Our data are consistent with the following model for generation of the deletion: (i) the formation of an intrastrand hairpin, and (ii) polymerization across the base of the hairpin, thus deleting the nucleotide sequence in the hairpin. Furthermore, we show that the deletion occurs mainly during synthesis of the (-)DNA strand. Our observations suggest that similar artifacts may occur in other sequences containing stable secondary structures.