Thermus polymerase

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

Taq DNA polymerase from Thermus aquaticus) has made it unnecessary to add fresh enzyme for each round of synthesis. Because the amount of target DNA theoretically doubles each round, 25 rounds would increase its concentration about 33 million times. In practice, the increase is actually more like a million times, which is more than ample for gene isolation. Thus, starting with a tiny... 

To overcome this shortcoming, biologists turned to an enzyme that could survive the PCR hot cycle. They replaced the original heat-sensitive enzyme with DNA-polymerase from Thermus acqua-ticus, the Yellowstone extremophile. The new enzyme is unscathed... 

Taq polymerase is a thermostable DNA polymerase which was originally isolated from the bacterium Thermus aquaticus, which lives in hot springs. 

RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

Several of the enzymes involved in the processes of repheating, transcription and reverse transcription are available commercially and are used by molecular biologists in the manipulation of nucleic acids. One of the most important of these is Taq polymerase (Taq), which is a thermostable DNA polymerase named after the thermophihe bacterium Thermus aquaticus from which it was originally isolated. This enzyme is especially important, as it is central to the technique known as PCR, which allows sophisticated, targeted in vitro amplification and manipulation of sections of DNA or RNA. DNA... 

The sequence of manipulations in the method is presented in Figure 3.25. An initial problem with the method was that, since the temperature used to separate the strands is about 90 °C, repetitive separation resulted in inactivation of the polymerase, so that fresh enzyme needed to be added for each cycle. The problem was solved by using a DNA polymerase extracted from the organism Thermus aquaticus, which lives in hot springs, so that the enzyme is stable at the high temperature needed to separate the strands. 

PCR makes use of the heat-stable enzyme DNA polymerase from the bacterium Thermus aquaticus and its ability to synthesize complementary strands of DNA when supplied with the necessary deoxyribonu-cleoside triphosphates. We have already looked at the chemistry of DNA replication (see Section 14.2.2), and this process is exactly the same, though it is carried out in the laboratory and has been automated. 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

FIGURE 26-4 Structure of the RNA polymerase holoenzyme of the bacterium Thermus aquaticus. (Derived from PDB ID 1 IW7.)The overall structure of this enzyme is very similar to that of the E. coli RNA polymerase no DNA or RNA is shown here. The j3 subunit is in gray, the j3 subunit is white the two a subunits are different shades of red the to subunit is yellow the cr subunit is orange. The image on the left is oriented as in Figure 26-6. When the structure is rotated 180° about the y axis (right) the small to subunit is visible. 

Other Class A polymerases. The Thermus aquati-cus (Taq) polymerase is best known for its widespread use in the polymerase chain reaction (PCR Fig. 5-47). Like E. coli I the enzyme is a large multidomain protein. The structure of the catalytic domains of the two enzymes are nearly identical, but the Taq polymerase has poor 3 -5 editing activity.276 The enzyme has been carefully engineered to improve its characteristics for use in the PCR reaction.277... 

Figure 28-4 (A) Hypothetical structure of a "transcription bubble" formed by an RNA polymerase. Shown is a double-stranded length of DNA with the unwound bubble in the center. This contains a short DNA-RNA hybrid helix formed by the growing mRNA. The DNA double helix is undergoing separation at point A as is the hybrid helix at point B. NTP is the ribonucleotide triphosphate substrate. See Yager and von Hippel.71 (B) Stereoscopic view of the structure of RNA polymerase from Thermus aquaticus in a complex with a promoter DNA. Included are the al, all, (0, (3, P, and a subunits. However, the a C-terminal domains have been omitted. The template (t) strand passes through a tunnel, which is formed by the P and P subunits and two of the structural domains of the a subunit. The nontemplate (nt) strand follows a different path. The position of the -10, -35, and UP elements of the DNA are marked. From Murakami et al.33d Courtesy of Seth A. Darst.

Tunis. M.A., K.B. Myambo, D.H. Gelfand and M.A. Brow 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85 9436-9440. 

Section 4.3), which had to be replenished at every new cycle, the application of heat-stable polymerases from organisms such as Thermus aquations (Taq polymerase), Pyrococcus woesii (Pwo polymerase), Pyrococcus juriosus (Pfu polymerase), or Thermococcus litoralis (Vent polymerase) facilitated automation of the thermal cycling process. 

Korolev, S., Nayal, M., Barnes, W. M., Di Cera, E., and Waksman, G. (1995). Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5 A resolution Structural basis for thermostability. Proc. Natl. Acad. Sd. USA 92, 9264-9268. 

Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996). Random mutagenesis of Thermus aquaticus DNA polymerase I Concordance of immutable sites in rhtowith the crystal structure. Proc. Natl. Acad. Sci. USA 93, 9670-9675. 

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