Indian Journal of Science and Technology

Abstract

Objectives: To analyze and compare the Multi-subunit (MSU) DNA dependent RNA Polymerases (RNAPs) of eubacteria and chloroplasts and to identify the extent of conservations between them with special reference to the active sites. Methods: The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from eubacteria and chloroplasts. The conserved motifs identified by the bioinformatics analysis and the biochemical and Sitedirected Mutagenesis (SDM) experiments and X-ray crystallographic analysis data available on the eubacterial MSU RNAPs are used to derive and analyze active site regions of the MSU RNAPs of chloroplasts. Findings: 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 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. Applications: Analysis of MSU RNAPs of chloroplasts assumes greater importance as it is the one that transcribes the foreign genes in chloroplast transformation experiments.

Keywords: Chloroplast Multi-subunit RNA Polymerases, Distance Conservations, Eubacterial RNA Polymerases, Metal Binding Regions, Multi-subunit RNA Polymerases, Plastid Encoded Polymerases, Polymerase Active Sites