Thermus aquaticus

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... 

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

Tanner, J J., R.M. Hecht, and K.L. Krause. 1996. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceralde-hyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. Biochemistry 35 2597-2609. 

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. 

Gihring, T. M., Druschel, G. K., McCleskey, R. B., Hamers, R. J. Banfield, J. F. 2001. Rapid arsenite oxidation by Thermus aquaticus and Thermus thermophilus Field and laboratory investigations. Environmental Science and Technology, 35, 3857-3862. 

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. 

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. 

Rather similar ribonucleotide reductases have been isolated from the thermophile, Thermus aquaticus (MW = 80 000) and Anabaena (a blue-green alga) (MW=72 000). The latter enzyme has an absolute requirement for divalent metal cations. The diphosphate reductase from Corynebacterium has a molecular weight of 200 000 and is made up of two subunits. Other enzymes appear to have tetrameric structures.817... 

Kjeldgaard, M., Nissen, P., Thirup, S., and Nyborg, J. (1993). The crystal structure of elongation factor EF-Tu from thermus aquaticus in the GTP conformation. Structure 1, 35-50. 

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