The
key role of thermus aquaticus
thermostability in modern biotechnology
Original Article: Brumm, P. J., Monsma, S., Keough, B., Jasinovica, S., Ferguson, E., Schoenfeld, T., & ... Mead, D. A. 2015. Complete Genome Sequence of Thermus aquaticus Y51MC23. Plos ONE, 10(10), 1-30.
Post by: Jackie Azelby
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| Fig. 1 Morning Glory Pool is a hot spring in the Upper Geyser Basin of Yellowstone Nation Park in the United States |
So I’m
sure you’ve heard of extreme sports and extreme couponing, however, when it
comes to extreme living, there are few organisms on this planet that thrive in
conditions more intense than the bacteria Thermus
aquaticus. Calling geothermal springs
and hydrothermal vents home (Fig. 1), T. aquaticus is noted for its thermophilic properties that allow it to survive at
very high temperatures,
ranging from 55-100 degrees Celsius. T. aquaticus
is a
rod-shaped gram-negative, chemoheterotrophic bacterium, meaning it acquires
energy and nutrients from chemicals and organic compounds, and inhabit waters
with a pH ranging from 5-9 (Fig 2 and 3).
 |
| Fig 2 Microscopic images of T. aquaticus structure |
 |
| Fig. 3 Contrast Microscopic images of T. aquaticus structur |
The
survival of this unique bacterium in such extreme environments can be
attributed to the evolutionary success of its thermostable enzymes, many of
which have become key players in the advancement of modern science and
biotechnology. Many of these enzymes, including DNA ligase, NADH oxidase, and Taq
1 restriction enzyme, have been isolated for use in high temperature molecular
biology applications. However, the most notable thermostable enzyme isolated
from T. aquaticus is its DNA
polymerase, which is officially referred to as Taq Polymerase.
 |
| Fig. 4 The structure of Taq Polymerase labeled with the finger, thumb, and palm structures of the polymerization region, 3'-5' exonuclease and 5' nuclease |
You
can think of Taq polymerase as a matchmaker that moves its way along the
template DNA strand, introducing each nucleotide to its specific complimentary
pair. Taq polymerase assembles DNA by pairing nucleotides in the 3’-5’
direction. This enzyme is made up of an N-terminal 5’-3’ exonuclease domain and
a Klentaq1 domain, which is further subdivided into finger, palm, and thumb
domains. The coordination of these domains is necessary for optimal
functionality of Taq polymerase’s matchmaking activities. The ability of Taq Polymerase to participate
in DNA replication at high temperatures with increased accuracy prompted its
complete replacement of E. Coli DNA
Polymerase I in Polymerase Chain Reaction (PCR), a
technology used for amplifying up to billions of copies of a short DNA sequences.
The advancement of PCR with Taq Polymerase has transformed PCR’s efficiency and
has enabled scientists to answer many biological questions in biomedical
science, ecological conservation, forensics, and beyond. Who would have thought
that this little bacterium could have such a huge impact!
 |
| Fig. 5 Diagram of Taq Polymerase's role in Polymerase Chain Reaction process |
Many
scientists have focused their research of Thermus
aquaticus on understanding the kinetics behind the protein stability of Taq
polymerase during DNA catalysis. One paper that explores the kinetics of Taq
Polymerase during DNA catalysis is titled “Conformational Dynamics of Thermus
aquaticus DNA Polymerase I during Catalysis”, written by Cuiling Xu, Brian A.
Maxwell, and Zucai Suo (2014). Previous research has established that a
pre-catalytic open-to-close conformational change of
the Finger domain takes place during nucleotide binding, however little
research has been conducted concerning the other subunits. The purpose of this
research was to better understand the specific motions of all Taq Polymerase
subunits and other DNA polymerases during nucleotide binding and incorporation
by comparing the conformational dynamics of full length Taq Polymerase to
truncated version consisting of only the DNA substrate. The authors used
stopped-flow Förster resonance energy transfer (FRET), a phenomenon that takes place between two fluorescent
molecules where energy is transmitted from an electronic excited state of one
molecule to the ground state of another, to evaluate the global conformational
dynamics of the enzyme. They also used a mutant of Taq Polymerase that
contained a de novo disulfide bond
between the Finger and Thumb domains to measure how limiting protein
flexibility would affect DNA polymerization.
 |
| Fig 6. The changes in FRET produced by DNA and DNA nucleotide binding measured using steady state fluorescence spectra of the extendable S-1 DNA substrate. Addition of the enzyme to the DNA substrate (black line) resulted in a decrease in donor emission at 517nm and in increase in acceptor fluorescences at 617nm, indicating and efficient FRET pair (red line). There was an increase in FRET of a DNA binding to a different nucleotide most likely due to translocation of DNA polymerase by one base pair along the DNA (green line). |
The results of FRET
testing showed that there is a global conformational change that is not only
limited the finger domain during nucleotide incorporation catalyzed by Taq
Polymerase, but rather takes place throughout all five subunits (Fig. 6). The
similarity of kinetic rates among all the domains suggest that the mechanistic
steps of conformational change are analogous between them, however the global
conformational transition occurs more quickly in the truncated form of Taq
Polymerase lacking the N-terminus compared to that of the full length version of the enzyme (Table 1). The disulfide Taq
Polymerase mutant could essentially lock
the enzyme in a position similar to a closed conformation, however, the results
suggest that this does not completely inhibit polymerization, but rather
reduces the enzyme activity compared to that of the wild-type.
 |
| Table 1: FRET efficiency between acceptor and donor on all domains of Taq Polymerase. Indicates TaqPol underwent a global conformational change during DNA and nucleotide binding. |
This
study has provided greater insight into the kinetic mechanisms of the catalysis
of DNA synthesis by the most widely used enzyme in high temperature
biotechnology applications. DNA polymerases are essential for cellular
replication and repair. As a result, the findings from this study may provide
insight into problems associated with enzymatic function that causes it to be
inhibited or altered. Better understanding of this enzyme’s unique thermostable
properties could, in turn, further improve and expand its application in the
field of biotechnology. This research could also be used to evaluate other
types of thermophiles and their potential uses in biotechnological fields.
Links
Quick
Facts on Taq Polymerase
Taq
Polymerase in Polymerase Chain Reaction
·
https://www.youtube.com/watch?v=iQsu3Kz9NYo
Literature:
Brumm, P. J., Monsma, S., Keough, B., Jasinovica, S., Ferguson,
E., Schoenfeld, T., & ... Mead, D. A. 2015. Complete Genome Sequence of
Thermus aquaticus Y51MC23. Plos ONE, 10(10), 1-30.
Erlich
HA, editor. PCR Technology: Principles and Applications for DNA Amplification.
1992. New York (NY): WH Freeman and Company; 246 p.
Xu, C., Maxwell, B., & Suo, Z.,
2014. Conformational Dynamics of Thermus aquaticus DNA Polymerase I during
Catalysis. Journal of Molecular Biology, 2901-2917.
References:
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