Måns Ehrenberg

Positive allosteric feedback regulation of the stringent response enzyme RelA by its product.

During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome‐dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency.

Initiation of bacterial protein synthesis with wild type and mutated variants of initiation factor 2

In-frame translation of code triplets (codons) of mRNAs into the peptide chains that define the cell’s proteome requires accurate positioning of the mRNA start codon in the ribosomal P site during initiation of protein synthesis. Initiation in bacteria begins on the small (30S) ribosomal subunit. It is generated through dissociation of the bacterial ribosome into its small and large (50S) subunits. Ribosome dissociation is promoted by ribosomal recycling factor, RRF, and elongation factor G (EF-G) (Pavlov et al., 1997; Karimi et al., 1999; Peske et al., 2005; Pavlov et al., 2008; Savelsbergh et al., 2009) following termination of protein synthesis by a class-1 release factor (RF1/2) (Freistroffer et al., 2000) and RF1/2 recycling by the G-protein RF3 (Freistroffer et al., 1997; Zavialov et al., 2001).

A Novel Mechanism for Activator-Controlled Initiation of DNA Replication that Resolves the Auto-regulation Sequestration Paradox

For bacterial genes to be inherited to the next bacterial generation, the gene containing DNA sequences must be duplicated before cell division so that each daughter cell contains a complete set of genes. The duplication process is called DNA replication and it starts at one defined site on the DNA molecule called the origin of replication (oriC) [1]. In addition to chromosomal DNA, bacteria often also contain plasmid DNA. Plasmids are extra-chromosomal DNA molecules carrying genes that increase the fitness of their host in certain environments, with genes encoding antibiotic resistance as a notorious example [2]. The chromosome is found at a low per cell copy number and initiation of replication takes place synchronously once every cell generation [3,4], while many plasmids exist at a high copy number and replication initiates asynchronously, throughout the cell generation [5]. In this chapter we present a novel mechanism for the control of initiation of replication, where one type of molecule may activate a round of replication by binding to the origin of replication and also regulate its own synthesis accurately. This mechanism of regulating the initiation of replication also offers a novel solution to the so-called auto-regulation sequestration paradox, i.e. how a molecule sequestered by binding to DNA may at the same time accurately regulate its own synthesis [6]. The novel regulatory mechanism is inspired by the molecular set-up of the replication control of the chromosome in the bacterium Escherichia coli and is here transferred into a plasmid model. This allows us to illustrate principles of replication control in a simple way and to put the novel mechanism into the context of a previous analysis of plasmids regulated by inhibitor-dilution copy number control [7]. We analyze factors important for a sensitive response of the replication initiation rate to changes in plasmid concentration in an asynchronous model and discover a novel mechanism for creating a high sensitivity. We further relate sensitivity to initiation synchrony in a synchronous model. Finally, we discuss the relevance of these findings for the control of chromosomal replication in bacteria.