Transcription terminators with context-dependent promoter and terminator activities

Abstract

A dogma of RNA synthesis is that promoter activity and termination are unrelated processes governed by completely different DNA sequences. Serendipitously, we find that two class II terminators for bacteriophage T7 RNA polymerase (RNAP), the natural T7 concatemer junction (CJ) and the artificial vesicular stomatitis virus (VSV) terminator, are context-dependent strong promoters for E. coli RNAP. However, the third class II member, the artificial human preproparathyroid hormone (PTH) terminator, is not a promoter. Transcription start sites and mutagenesis for CJ and VSV reveal a {sigma}70 extended TGn promoter motif that is lacking in PTH. Furthermore, results resolve the prior paradox of class II termination apparently occurring only in vitro, not in vivo, enabling demonstration of class II termination in vivo by both T7 and E. coli RNAPs. In addition, engineering of T7 RNAP increases its efficiency of class II termination in vivo. Given that prokaryotic synthetic biology is reliant on class I (hairpin) terminators, with T{Phi} used almost exclusively in constructs for T7 RNAP, recombination is an issue in large constructs. Thus, small class II terminators, now better understood with respect to terminator and promoter activities in the context of various sequences and RNAPs, extends the synthetic biology toolkit.