Forster, A. C. | Search bioRxiv

bioRxiv2026

Transcription terminators can be strong promoters

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.

molecular biology↗

bioRxiv2024

Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally-important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the "critical region" of the peptidyl transferase center (PTC) in 23S rRNA that exhibited only a minor growth defect at 37{degrees}C (although major at 20{degrees}C). However, our combined KO of modification enzymes RluC and RlmE resulted in conditional lethality (at 20{degrees}C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20 and 37{degrees}C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Rates of protein synthesis in vivo, as judged by the kinetics of {beta}-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37{degrees}C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

biochemistry↗