Aim: To analyze and compare the active sites of the multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eubacteria and plant chloroplasts and find out the conserved motifs, metalbinding sites and catalytic regions and propose a plausible mechanism of action for the chloroplast MSU RNAPs using Zea mays enzyme 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 these enzymes were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various bacterial and chloroplast enzyme 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 both the sources showed many highly conserved motifs among them. The possible catalytic regions in the catalytic subunits β and β’ of eubacteria and their counterparts, viz. β, β’ and in chloroplasts RNAPs consist of an absolutely conserved catalytic amino acid R, in contrast to a K as reported for DNA polymerases and single subunit (SSU) RNAPs. Besides, the invariant ‘gatekeeper/DNA template binding’ YG pair is also found to be absolutely conserved in the MSU RNAPs of chloroplasts, as reported in SSU, MSU RNAPs and DNA polymerases. The eubacterial β, the initiation subunit, is highly homologous to β subunit of chloroplast MSU RNAPs, i.e., the eubacterial and chloroplast β subunits exhibit very similar active site motifs, catalytic regions and distance conservations between the template binding YG pair and the catalytic R. However, the bacterial β’ elongation subunit is not completely similar to the β’ elongation subunit of chloroplasts, but partly similar to the β’and β’’ subunits of chloroplast RNAPs. Interestingly, MSA analysis shows that the active sites are, in fact, shared between β’ and β’’ in the MSU RNAPs of chloroplasts, i.e., the metal-binding site is found in the β’ subunit whereas the catalytic regions are located in β’’ subunit of chloroplast MSU RNAPs. Another interesting finding is, in the elongation subunits, i.e., in the eubacterial β’ and the chloroplast β’’ catalytic subunits, the proposed catalytic R is placed at double the distance, i.e., -16 amino acids downstream from the YG pair, in contrast to SSU RNAPs and DNA polymerases where the distance is only ~8 amino acids downstream from the YG pair. An invariant Zn2+ binding motif reported in the eubacterial elongation subunit, viz., β’ is found in the β’’ subunits of chloroplasts. The catalytic R, along with the Zn binding motif is shifted towards the N-terminal in the elongation subunit of PEP. Conclusions: MSA have shown that in both the MSU RNAPs of eubacteria and chloroplasts, the active sites, catalytic amino acids and metal-binding regions are absolutely conserved both in the initiation and elongation subunits. Therefore, it is suggested that the MSU RNAPs of chloroplasts may also follow very similar polymerization and proof-reading mechanisms as proposed for eubacteria. MSA data and the available experimental data show that both the eubacterial and chloroplast MSU RNAPs would have possibly evolved from a common ancestor.
Dr. Peramachi Palanivelu
Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India.
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