Eukaryotic Multi-subunit DNA Dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Aim: To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs)
of eukaryotic organisms and find out conserved motifs, metal-binding sites and catalytic regions and
propose a plausible mechanism of action for these complex eukaryotic MSU RNAPs, using yeast
(Saccharomyces cerevisiae) RNAP II, as a model enzyme.
Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic
data were analyzed.
Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India,
between 2007- 2013.
Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray
crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was
used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic
sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already
available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes
were used to confirm the possible amino acids involved in the active sites and catalysis.
Results: Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a
large number of highly conserved motifs among them. Possible catalytic regions in the catalytic
subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely
conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit
(SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported
in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in
eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the
human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation
subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance
conservations between the template binding YG/KG pair and the catalytic R. In the eukaryotic
initiation subunits, the proposed catalytic R is placed at the -9th position from the YG/KG pair and an
invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP)
selection as reported for SSU RNAPs (viral family) and DNA polymerases.
Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to
the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regions are highly conserved,
and the metal-binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes,
the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to
the prokaryotic β’ subunits, in the eukaryotic Rpb1 elongation subunits also, the proposed catalytic R
is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU
RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG
pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6th position are
proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the
elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three
completely conserved Cs is also highly conserved in the eukaryotic elongation subunits also. A plausible proof-reading mechanism during the elongation process is also proposed based on the MSA
and experimental data. Another important difference is that the catalytic region is placed very close to
the N-terminal region in eukaryotes.
Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of
eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance
conservation in the initiation and elongation subunits. An invariant Zn2+ binding motif, found in the
Rpb1 elongation subunits is proposed to participate in the proof-reading function. Differences in the
active sites of bacterial and human RNA polymerases may pave the way for the design of new and
effective drugs for many bacterial infections, including the multidrug-resistant strains which are a
global crisis at present.