Indian Journal of Science and Technology

Abstract

Objectives: To analyze the Multi-Subunit (MSU) DNA dependent RNA polymerases of eubacteria and identify conserved motifs, active site regions among them and propose a plausible mechanism of action for these enzymes, using the E. coli RNA polymerase as a model system. Methods: The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNA polymerases from eubacteria. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and Site-Directed Mutagenesis (SDM) experiments and X-ray crystallographic analysis on these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Findings: Multiple Sequence Alignment (MSA) of eubacterial RNA polymerases from different bacterial sources showed only a very few highly conserved motifs among them. Possible catalytic regions in the catalytic subunits β and β‘ (in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that is reported for DNA polymerases and single subunit (SSU)DNA dependent RNA polymerases. In addition to, the invariant ‘gatekeeper/DNA template binding’ YG pair is also found in all the MSU RNA polymerases as found in all the SSU RNA polymerases and DNA polymerases. The eubacterial βsubunits are very similar in theiractive sites, catalytic regions and distance conservations between the template binding YG pair and the catalytic R, i.e., they are placed at 7 amino acids length. An invariant R is placed here at -6th position from the catalytic R, similar to SSU RNA polymerases and DNA polymerases which is proposed to play an important role in NTP selection. In the eubacterial β’ catalytic subunits, the proposed catalytic R is placed double the distance, i.e., -16 amino acids downstream from the YG pair unlike in the SSU RNA polymerasesand DNA polymerases where the distance is only 8th amino acids downstream from the YG pair. The two Ds in the completely conserved metal binding region could act as the charge shielder and orient the α-phosphates of incoming NTPs for reaction at the 3’-OH growing end. Based on the sequence analysis, a model is proposed for the initiation and elongation steps in the transcription process and a plausible mechanism of action. Applications: A comparative analysis of the bacterial and human RNA polymerases for their transcription mechanism will pave way for design new and effective drugs for many bacterial infections, including the antibiotic resistance.

Keywords: Bacterial RNA Polymerases, Clustal Omega, Conserved Motifs, Multi-Subunit RNA Polymerases, Polymerase Active Sites, Polymerization Mechanism, Proofreading