A two-part res­ur­rec­tion tale (2|2)

by Christoph

In continuation of the first part of the tale: what is the "resurrection" in the title about? A bit of nostalgia, as I will "resurrect" a story that is based on research from ~30 years ago (and today mainly known by dedicated plasmid specialists, if at all). A simple but vexing question bothered researchers studying conjugation: how could genes transferred from a conjugative plasmid into a recipient cell be expressed so quickly, that is, within minutes, as their time-course experiments showed? That is, well before the plasmid transfer was complete and the transferred DNA still single-stranded.

Time‑staggered expression of "early," "middle" and "late" genes was already well known back then from bacteriophages, for example from E. coli phage T7. The segment of the linear T7 chromosome that first enters the host cytoplasm upon infection carries the "early" genes that are transcribed by the host RNA polymerase. This segment carries seven genes, among them the gene for a restriction inhibitor (gp0.3) and the gene for T7 RNA polymerase (gp1) (see here for a concise explainer). Yet, T7 DNA is double‑stranded and the three major early promoters are readily recognized by the host RNA polymerase. Could single‑stranded DNA trigger transcription in the case of conjugative plasmids?

Figure 3. Proposed secondary structure of ssFrpo and a possible mode of recognition by RNA polymerase. Nucleotides protected from DNase I digestion are indicated by gray (for RNA polymerase) and by striped (SSB) boxes. Gray and black arrows indicate, respectively, the position of the major 59 end of the primer RNA synthesized in the purified system and that of the transition site from RNA to DNA in the crude extract. The arrows indicate the direction of transcription/DNA chain elongation. The residues sensitive to P1 nuclease near the top of the stem are circled. Putative −10 and −35 sequences are boxed. Source

The answer is: yes, it can, but there's a twist. In 1997, Hisao Masai and Ken-ichi Arai from the University of Tokyo, Japan, published in Cell their finding of "Frpo : A Novel Single-Stranded DNA Promoter for Transcription and for Primer RNA Synthesis of DNA Replication." The first part of the title of their paper is important here, but their study started with the latter: during characterization of ssi, the genetically identified single‑strand replication origin of the (conjugative) F plasmid, they found a novel priming signal for DNA replication. Primer RNA synthesis on ssi is mediated by RNA polymerase and, therefore, they designated it Frpo. In the presence of SSB, RNA polymerase efficiently initiates transcription at a specific site on ssDNA containing Frpo, and RNAs are elongated into DNA chains by DNA Pol III holoenzyme. For the first part of the title: Frpo appears to also provide transcripts for downstream open reading frames (ORFs) of the F plasmid since the transcription initiation site on Frpo identified in vitro is identical to that previously localized in vivo for these ORFs (whose identity was not known then but is now. One of them, PsiB, is an inhibitor of the SOS response, that is, an "anti-defense" gene). Hallmark of the single‑stranded Frpo promoter – and the "twist" I mentioned earlier – is an extended foldback or stem-loop structure that contains, within the double‑stranded DNA stem, the canonical –35 and –10 elements of bacterial promoters (Figure 3). The observation that RNA polymerase holoenzyme (αββ'ω+σ⁷⁰) is active in in vitro transcription from Frpo while core RNA polymerase (αββ'ω) is not supports the assignment of the –35 and –10 elements since the σ⁷⁰ subunit confers promoter specificity to RNA polymerase. On double‑stranded DNA, Frpo is transcriptionally inactive in vitro, but active on denatured, that is, single-stranded DNA covered with single strand‑binding protein (SSB), which is thought to stabilize the stem-loop structure (and certainly prevents re‑annealing of the DNA strands).

Frpo-type promoters were detected not much later by Bates et al. (1999) in the ssi region of the conjugative plasmid ColIb-P9 (see here). Today we know – not least from the study by Samuel & Burstein (2023) – that ssDNA promoters of the Frpo type are abundant in DNA elements such as plasmids (and phages) that are transiently single-stranded. To my knowledge, Frpo-type promoters have not yet been found in Archaea or Eukaryotes. For me, it is a joy to see that a branch of experimental research that initially seemed to be rather eccentric, almost wacky, and clearly more of a niche pursuit, has in the long run contributed to a better understanding of widespread processes.

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