Single Subunit RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism | Chapter 01 | Advances and Trends in Biotechnology and Genetics Vol. 1
Aim:
To analyze various single subunit DNA dependent RNA polymerases and identify
conserved motifs, active site regions among them and propose a plausible
mechanism of action for these polymerases using the T7 RNA polymerase as a
model system.
Study
Design: Bioinformatics, Biochemical, Site-directed
mutagenesis and X-ray crystallographic data were analyzed.
Place
and Duration of Study: Department of Molecular Microbiology,
School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India,
from 2010 to 2013.
Methodology:
The advanced version of Clustal Omega was used for protein sequence analysis of
various SSU DNA dependent RNA polymerases from viruses, mitochondria and
chloroplasts. Along with the conserved motifs identified by the bioinformatics analysis
and with the data obtained by X-ray crystallographic, biochemical and site-directed
mutagenesis (SDM) were also used to confirm the possible amino acids involved
in the active sites and catalysis of these RNA polymerases.
Results:
Multiple sequence analyses of various single subunit (SSU) DNA dependent RNA polymerases
from different sources showed only a few highly conserved motifs among them,
except chloroplast RNA polymerases where a large number of highly conserved
motifs were found. Possible catalytic regions in all these polymerases consist
of a highly conserved amino acid K and a ‘gatekeeper’ YG pair. In addition to,
these polymerases also use an invariant R at the -4 position from the YG pair
and an invariant S/T, adjacent to the YG pair. Furthermore, two highly
conserved Ds are implicated in the metal-binding site
and thus might participate in the catalytic process. The YG pair appears to be
specific for DNA templates as it is not reported in RNA dependent RNA
polymerases.
Conclusion:
The highly conserved amino acid K, the ‘gatekeeper’ YG pair and an invariant R
which are reported in all DNA polymerases, are also found in these DNA
dependent RNA polymerases. Therefore, these RNA polymerases might be using the
same catalytic mechanism as DNA polymerases. The catalytic amino acid K could
act as the proton abstractor and generate the necessary nucleophile at the
3’-OH and the YG pair, R and the S/T might involve in the template binding and
selection of nucleoside triphosphates (NTPs) for polymerization reactions. The
two highly conserved Ds could act as the ‘NTP charge shielder’ and orient the
alpha phosphate of incoming NTPs for the reaction at the 3’-OH growing end.
Author(s) Details
Dr. Peramachi Palanivelu
Department of Molecular
Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai –625
021, India (Retd.).
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