Características del sistema de desinfección

All known DNA and RNA polymerases employ two divalent metal ions in order to perform the phosphoryl transfer reaction (Brautigam and Steitz 1998). One metal ion is chelated by active site residues and is responsible for the reduction of the 3’OH group of the substrate nucleic acid chain (DNA or RNA). The other metal ion is transferred into the catalytic site bound to the phosphate moiety of the incoming NTP and is used in the nucleophilic attack of the α-phosphorous atom.

Previous work on RNA dependent RNA polymerases has shown that quenching with EDTA or HCl leads to the elongation reaction being quenched at different stages of incorporation (Arnold and Cameron 2004; Arnold, Gohara et al. 2004). The difference between EDTA and HCl quench is due to how each molecule quenches the reaction. EDTA quenches the reaction by chelating the Mg+2 that is associated with the incoming NTP; NTPs are unable to be incorporated because one of the two required Mg+2 is no longer available for use in catalysis. However, elongation complexes that have an NTP already bound in the active site prior to the addition of EDTA are immune to quenching because EDTA cannot chelate the Mg+2 ion; therefore, the reaction is able to continue towards completion. HCl quenches the incorporation reaction instantaneously upon addition by denaturing the enzyme, which demonstrates the time of bond formation.

Because RNAP uses the divalent metal ion system to perform its catalytic functions, quenching the reaction with EDTA or HCl will lead to stopping of the elongation reaction at different stages.

4.1.2 Quenching with HCl or EDTA Alters AMP Incorporation

Previous work by Burton and coworkers has shown that quenching elongation reactions with EDTA and HCl alters the kinetics of nucleotide addition in RNAPII (Gong, Zhang et al. 2005; Zhang, Zobeck et al. 2005). Due to these previously published results, we were interested in examining the effect of pre-incubating the i+2 nucleotide (ATP) on the rates of incorporation of the i+1 nucleotide (CMP), and the i+2 nucleotide (AMP) when complexes were quenched with HCl instead of EDTA. To that end, we performed experiments in which SECs were pre-incubated with either 10μM prior to the addition of CTP (100μM) and then quenched the reaction by the addition of 1M HCl. Simultaneous addition experiments were done as controls using the same NTP concentrations as the pre-incubation experiments (Figure 4.1).

The data for AMP incorporation exhibits biphasic kinetics with a fast phase followed by a slow phase. The biphasic kinetics are readily apparent in the simultaneous addition experiments. The results for both pre-incubation simultaneous addition

experiments show a marked decrease in the extent of complexes in the fast phase for AMP incorporation compared to reactions quenched with EDTA (Figure 4.2A and 4.2B). Interesting, there is a ~40% reduction in the percentage of complexes in the fast chase for both the pre-incubation and simultaneous addition experiments when quenched with HCl instead of EDTA. This result means that, of the complexes in which ATP is isomerized

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Figure 4.1 Experimental Design to Test for Differences in Quench Conditions. Purified SECs are split into two groups; one in which 10μM ATP is added to the complexes prior to the addition of 100μM CTP via rapid quench, and one in which 100μM CTP and 10μM ATP are added simultaneously by rapid quench techniques. The reactions are either quenched with 0.5M EDTA or with 1M HCl.

Figure 4.2A

Figure 4.2B

Figure 4.2 Quenching Reactions with EDTA or HCl Alters the Extents of AMP Incorporation.

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Figure 4.2 Quenching Reactions with EDTA or HCl Alters the kinetics AMP Incorporation. All EDTA quenched reactions are designated in black while all HCl quenched reactions are designated in red. Fits for CMP incorporation data (25+) are solid while curve fits for AMP incorporation data (26+) are dashed.

A. Plots are for pre-incubation experiments. The extent and rate of AMP incorporation is reduced by quenching with HCl when compared to experiments using EDTA as the quench. The EDTA quench data are presented in Chapter 2.

B. Plots are for the simultaneous addition experiments. The extent and rate of AMP incorporation is reduced by quenching with HCl when compared to experiments using EDTA as the quench. The EDTA quench data are presented in Chapter 2.

into the catalytic site, only half of them incorporate AMP prior to being quenched with HCl. This result is in stark contrast to what is observed for RNAPII(Gong, Zhang et al.2005).

Analogous experiments to the simultaneous addition experiments were carried out with human RNAPII. When quenched with HCl, RNAPII elongation complexes no longer exhibited biphasic kinetics. Instead, the fast phase for NMP incorporation was completely abolished, leaving complexes only in the slow phase (Gong, Zhang et al. 2005; Zhang, Zobeck et al. 2005). Prokaryotic RNAP does not exhibit a complete disappearance of the fast phase. This suggests that binding of an NTP into the catalytic site is faster in prokaryotic RNAP compared to eukaryotic RNAPII.

The rate of AMP incorporation for the fast phase in the pre-incubation

experiments is significantly reduced in experiments in which reactions were quenched with HCl instead of EDTA. Specifically, there is a 49%±16% reduction in the rate of AMP when quenched with HCl. Given that quenching with HCl gives the rate of bond formation, then the ~50% reduction in rate is due to the rate of bond synthesis being ~50% slower than the rate of NTP binding into the active site (EDTA quench). The rate reduction is consistent with the reduction in the extent of AMP incorporation; if NTP incorporation is half as fast at the rate of NTP binding to the catalytic site, then only half of the complexes will incorporate an NTP. Additionally, there is a reduction in the rate of AMP incorporation for the simultaneous addition experiments; however, there is too much error in the measurements to be able to obtain an actual rate.