In order to find the functional amino acid residues of SP6 RNA polymerase that are potentially involved in transcription termination, we have developed a two vector system that allows for isolating the mutants of phage SP6 RNA polymerase with increased efficiency of termination at the SP6 terminator. Mutations were induced by carrying out the polymerase chain reaction under reduced fidelity condition in N-terminal region (amino acid 1 273) of the SP6 RNA polymerase. From this method, we identified three point mutants (M15L, M15S, and D117G). And we purified the proteolytically cleaved SP6 RNA polymerase in E. coli JM109.
Transcription termination efficiencies of these mutant polymerases over various terminators were measured in vitro with the purified polymerases. At one class of terminators including its own (SP6 terminator and rrnBT1 terminator site A) these mutant enzymes terminated transcription with a higher efficiency than the wild type RNA polymerase. On the other hand, they terminated transcription with a lower efficiency at the rrnBT1 termination site B. And these mutant polymerases were deficient in processivity on long DNA templates. Comparision of the binding products for wild and mutant RNA polymerases with non-specific RNA and SP6 terminator RNA revealed that mutant enzymes have reduced binding activities with non-specific RNA and SP6 terminator RNA, respectively. And the binding activities of mutant RNA polymerases with terminator RNA were lower than with non-specific RNA.
From this study, we suggest that increase of termination efficiency of mutant RNA polymerases at class I terminator is result from the increase of RNA release at termination site.