Variation of factor-independent transcription termination efficiency depending on transcribed sequences

Abstract

Bacteriophage T7 genome has one transcription terminator sequence for the phage-encoded RNA polymerase located downstream of the gene 10 and named Tφ. The factor-independent 41-bp terminator sequence along with its flanking sequence was cloned downstream of strong phage T7 promoters in various plasmids. Its transcription termination efficiency was measured as the molar ratio of terminated transcripts to total transcripts produced from in vitro transcription reactions containing only purified T7 RNA polymerase, DNA template and ribonucleotides under standard conditions. The measured efficiency varied 17% to 82% depending on the sequences within transcriptional unit, but it does not depend on parental plasmid sequences. The genomic sequence immediately downstream of the initiation site can yield a 21-nucleotide RNA hairpin structure and cause the high termination efficiency. However, replacement with another RNA hairpin-forming sequence at the same site reduced the efficiency substancially. Also the removal of genomic sequence dose not change the efficiency. Thus, RNA hairpin structure itself is not responsible for the high efficiency of pET-3. The transcription termination efficiency of Tφ is not determined by only initial transcribed region but by the entire sequence in a transcription unit.