Reduction of factor-independent transcription termination efficiency by trans-acting abortive transcript RNA

Abstract

Transcripts of pGEM3ZT has been proposed to cause termination efficiency reduction [Lee, J. T., and Kang, C., (1992) Biochem. Int. 26, 163-170]. By transcription with incomplete sets of nucleotides, abortive transcripts were produced exclusively. To test the trans-acting activity, T7 promoter was destructed by HinfI digestion, T7 RNA polymerase and HinfI were removed by phenol extraction, and then the abortive products were included in the second reaction with a second template and newly added polymerase. Abortive transcripts of pGEM3ZT produced with any set of nucleotides have the activity. pET3a showed trans-acting antitermination activity if an incomplete set of nucleotides was supplied. It seems that abortive transcripts are trans-acting antitermination factor. The sequence of pGEM3T is identical to pGEM3ZT except few sites in initially transcribed region but it does not show trans-acting antitermination activity. The aberration from consensus promoter sequence at +4 position makes pGEM3ZT produce remarkable amount of abortive transcripts. Termination of transcription by T7 RNA polymerase and SP6 RNA polymerase at rrnBT1 E. coli terminator and termination by SP6 RNA polymerase and T3 RNA polymerase at T7 terminator T$\phi$ were tested. Antitermination is not testricted to T7 RNA polymerase but rrnBT1 terminator is resstant to antitermination.