Other Polymerases

Pol II belongs to the family of multisubunit RNA polymerases, which also comprises the two other eukaryotic RNA polymerases, Pol I and Pol III. Pol I and Pol III are mainly responsible for synthesis of ribosomal RNA 

There are, however, minor differences on the enzymes surfaces caused by amino acid insertions and deletions. These differences are most likely responsible for conferring specificity toward the interaction with factors specific for Pol I, II, and III. In addition to the 12 subunits that are either identical or homologous, Pol I contains two specific subunits, A34.5 and A49, and Pol III contains a subcomplex of three specific subunits, called C82, C34, and C31, in yeast. The location of the two Pol I—specific subunits has been determined by electron microscopy and immunolabeling (Bischler et al., 2002). The Pol I subunit A49 binds to the top of the clamp, and subunit A34.5 is located near the jaws. The location of the specific C82/C34/C31 complex of Pol III can be inferred from subunit-subunit interaction studies (Ferri et al., 2000 Flores et al., 1999). These studies indicate that the specific subcomplex is located between the largest polymerase subunit and the Rpb4/7 complex counterpart C17/C25. The Cll subunit of Pol III contains a C-terminal domain that apparently corresponds structurally and functionally to domain III of TFIIS (Chedin et al., 1998 Kettenberger et al., 2003), which inserts into the polymerase pore. Thus, in Pol III, the RNA cleavage stimulatory activity is incorporated into a polymerase subunit, in contrast to Pol II, where it is provided by the additional factor TFIIS. 

Bacteria and archaea contain a single multisubunit RNA polymerase. X-ray crystallographic structures were determined of a bacterial RNA polymerase from Thermus aquaticus at 3.3-A resolution (Darst, 2001 Zhang et al., 1999). Comparison of this bacterial RNA polymerase structure 

Structures of multisubunit RNA polymerases are strikingly different from structures of polymerases of other families, such as the many single-subunit DNA and RNA polymerases. X-ray crystallography of 

Detailed structures are now available for the Pol II core enzyme in free form, in the form of a minimal elongation complex with bound nucleic acids, and in an inhibited form with bound a-amanitin. In addition, 

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This is a smaller, stable enzyme that has been highly purified. It is immunologically distinct from the other polymerases, indicating that it is not merely a subunit of the larger polymerases. Polymerase p is undoubtedly a repair enzyme. 

Polymerase for amplification. Pyrobest DNA Polymerase (TaKaRa BIO inc.) or PrimeSTAR DNA Polymerase (TaKaRa BIO INC.) are our recommendation. Other polymerases are also possible, but it is essential to select ones with extremely high fidelity. Buffers and dNTPs are usually supplied together with polymerases from manufactures. 

Recent kinetic work on RB69 polymerase (family B) and structural comparison between the RB69 polymerase and other polymerase families led to the postulation of a different initial binding event for dNTP (Yang et al, 2002a). The crystal structure of the closed ternary E p/t ddNTP complex of the RB69 polymerase (see Section VII) shows interactions between the fingers subdomain and the ddNTP s triphosphate moiety... 

In principle, the screening concepts introduced here could be applied to other polymerases, and other activity. As yet, however, the contexts of low polymerase fidelity or of tolerance versus non-natural substrates have not been sufficiently targeted. In the course of our studies, we tackled these problems and presented solutions for both, selecting, or screening, polymerase libraries. Thereby, we detected an error-prone polymerase variant and polymerase activity in the sole presence of sterically demanding substrates. 

Figure 13.1 General structural features for replicative and translesion polymerases. A structure of Dpo4 from S. solfataricus is shown to highlight general structural features associated DNA polymerases. The overall topology of polymerase subdomains has been likened to a right hand, as indicated in cartoon form. Three other polymerase...

DNA-directed DNA polymerase activity This activity replicates the minus strand DNA to yield dsDNA after viral RNA genome is degraded. The polymerase domain of HIV-1 RT shares structural similarity with other polymerases, presumably catalyzing the polymerization with the same reaction mechanism (Steitz, 1993 Steitz, 1998). 

Belguise-Valladier, P., Maki, H., Sekiguchi, M., and Fuchs, R. P. P. (1994). Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme Comparison with other polymerases. J. MoL Biol. 236, 151-164. 

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