Functional analysis of Methanocaldococcus jannaschii RNA polymerase A’ ‘bridge-helix’ using a fully automated high-throughput robotic system

Abstract

RNA polymerases (RNAPs) are the ‘engines’ of cellular transcriptional machineries, which are essential to life and highly conserved from bacteria to eukaryotes. Though crystal structures of both eukaryotic and bacterial RNAPs have been intensively studied, the relationships between structures and appropriate functions of such enzymes in eukaryotes remain unknown since there has not yet been possible to constitute any active eukaryotic RNAPs from recombinant subunits. The successfully assembled archaeal counterparts have provided an alternative approach to study the eukaryotic system due to not only the structural similarities between these enzymes but also the structural and functional similarities of their basal transcriptional machineries. ‘Bridge-helix’ is one of the most highly conserved structures near the catalytic site of RNAPs, which has been proposed to play an important role in coordinating the processing of nucleic acid substrates through the active center. 17 adjacent residues (mjA’-L814 to mjA’-R830) within the central portion of Methanocaldococcus jannaschii A’ ‘bridge-helix’ were chosen for a systematic high-throughput sitedirected mutagenesis approach using a novel robotic system. This robotic system is fully automated without any human interventions that may enormously reduce human errors and effectively increase the number of samples that could be processed in parallel. The results obtained from such high-throughput approach showed a wide spectrum of in vitro phenotypes ranging from complete loss of function to ‘superactivity’. According to the unexpected functional evidences obtained with the ‘superactive’ mutants, we propose a highly favorable kinked ‘bridge-helix’ conformation for the nucleotide addition cycle that has to be precisely localized in certain positions in order to increase the specific activity of RNAPs. The fact that no additive effects have been found so far in any of the ‘superactive’ double mutants suggests that various single amino-acid substitution ‘superactive’ mutants may affect the same process in a functionally overlapping and mutually independent manner.