Single Subunit RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

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.