Abstract

Nucleic acid synthesis is precisely controlled in living organisms by highly evolved protein enzymes. The remarkable fidelity of information transfer realized between template and product strands is the result of both the spatial selectivity of the polymerase active site for Watson–Crick base pairs at the point of nucleotide coupling and subsequent proof-reading mechanisms. In the absence of naturally derived polymerases, in vitro template-directed synthesis by means of chemically activated mononucleotides has proven remarkably inefficient and error-prone. Nevertheless, the spontaneous emergence of RNA polymers and their protein-free replication is frequently taken as a prerequisite for the hypothetical ‘RNA world’. We present two specific difficulties that face the de novo synthesis of RNA-like polymers in a prebiotic (enzyme-free) environment: nucleoside base selection and intramolecular strand cyclization. These two problems are inherent to the assumption that RNA formed de novo from pre-existing, chemically-activated mononucleotides in solution. As a possible resolution to these problems, we present arguments and experimental support for our hypothesis that small molecules (referred to as ‘molecular midwives’) and alternative backbone linkages (under equilibrium control) facilitated the emergence of the first RNA-like polymers of life.

A powerful strategy to translate DNA directly into synthetic polymers: A solid-phase synthetic strategy is applied to DNA-templated synthesis by using immobilized native DNA octamer S(dA_p)₈ as a template to direct the specific polymerization of synthetic DNA analogues (T)₁ and (T_N)₂ (see figure). The solid-supported template provides advantages in chemical amplification and purification.

The cover picture shows a representation of a ratchet model where the integration of multiple signals by the transmitter kinase VirA is achieved by xenognostic signal induced movement between three accessible homodimeric coiled-coil interfaces. It is this interface registry that activates the kinase domain, initiating lateral gene transfer from Agrobacterium tumefaciens to its eukaryotic host. Further details can be found in the article by Lynn and co-workers on p. 311 ff.

The transmembrane histidine kinase VirA is responsible for the recognition of information from several plant-derived xenognostic signals that control gene transfer between Agrobacterium tumefaciens and its eukaryotic host. As with other histidine autokinases, VirA appears to exist as a homodimer within the inner membrane of the bacterium. In this study, we identify the putative homodimeric coiled-coil-like motifs Helix TM2 (amino acids (aa) 259–288) and Helix C (aa 293–327) within the previously assigned signal input domain. The functional importance of these coiled-coil interactions in signal-mediated VirA activation is investigated by the construction of fusion proteins with the leucine zipper domain of the transcription factor GCN4. Replacement of the membrane-spanning and periplasmic domains of VirA with the GCN4 leucine zipper gave functional proteins with increased signal-induced vir gene expression. When the GCN4 fusion was used to conformationally bias the interface of the Helix C coiled coil, constitutively active chimeras were created. The activity of these constructs was dependent on the interface of the Helix C coiled coil, and a ratchet model is proposed in which VirA activation is achieved by signal-induced switching of the interfaces of the homodimer. Since VirA functions as a transducer and integrates various host cues indirectly, these data highlight its role as an “antenna” for the tumor-inducing (Ti) plasmid, able to monitor the host proteome so as to select for successful xenognostic signaling strategies.

Eine breit anwendbare Strategie zur direkten Übersetzung von DNA in synthetische Polymere wurde bei der Festphasensynthese unter Verwendung des immobilisierten nativen DNA-Oktamers S(dA_p)₈ als DNA-Templat zur Lenkung der spezifischen Polymerisation der synthetischen DNA-Analoga (T)₁ und (T_N)₂ verwendet (siehe Bild). Das festphasengebundene Templat hat praktische Vorteile bei der chemischen Amplifizierung und Reinigung.