In vitro transcription analysis by reconstituted cyanobacterial RNA polymerase: roles of group 1 and 2 sigma factors and a core subunit, RpoC2

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Abstract
The RNA polymerase (RNAP) core enzyme of cyanobacterium Synechocystis sp. strain PCC 6803 was reconstituted with overproduced recombinant subunits and purified with C-terminal histidine-tagged RpoA. The core enzyme with purified a sigma factor, SigA/SigD or SigB, allowed specific in vitro transcription from the light-inducible psbA2 or the dark-/heat-inducible lrtA/hspA promoters, respectively. Further analysis using a mutant psbA2 promoter revealed that the -35 hexamer of the promoter was essential for SigA but not SigD. Similar but distinct patterns of psbA2 transcription were found for two types of RNAP, cyanobacterial (alpha2betabeta'gamma) and E. coli (alpha2betabeta') core enzymes. Specific binding of PCC 6803 RpoC2 (beta') to E. coli core enzyme and its contribution to efficient psbA2 transcription by RNAP-SigA/D suggest that this subunit could confer an important role on the cyanobactrial RNAP. Differences in affinity and specificity among cyanobacterial sigma factors for the core enzyme and promoters were discussed.