Previous research has indicated that the growth rate-dependent regulation of Escherichia coli gnd expression involves the internal complementary sequence (ICS), a negative control site that lies within the 6-phosphogluconate dehydrogenase coding sequence. To determine whether the ICS acts as a transcriptional operator or attenuator, we measured ␤-galactosidase-specific activities in strains carrying gnd-lac operon and protein fusions containing or lacking the ICS. Whereas the presence of the ICS repressed ␤-galactosidase expression from a protein fusion by 5-fold during growth on acetate and by 2.5-fold during growth on glucose, it had no effect on ␤-galactosidase expression from an operon fusion. In vitro ribosome binding experiments employing the primer extension inhibition (toeprint) assay demonstrated that the presence of the ICS in gnd mRNA reduces both the maximum extent and the rate of ternary complex formation. Moreover, the effects of deletions scanning the ICS on in vivo gene expression were highly correlated with the effects of the deletions on ribosome binding in vitro. In addition, the distal end of the ICS element was found to contribute more to ICS function than did the proximal portion, which contains the complement to the Shine-Dalgarno sequence. Finally, RNA structure mapping experiments indicated that the presence of the ICS in gnd mRNA reduces the access of the nucleotides of the ribosome binding site to the single-strand-specific chemical reagents dimethyl sulfate and kethoxal. Taken together, these data support the hypothesis that the role of the ICS in the growth ratedependent regulation of gnd expression is to sequester the translation initiation region into a long-range mRNA secondary structure that blocks ribosome binding and thereby reduces the frequency of translation initiation.Growth rate-dependent regulation is a fundamental genetic regulatory process that coordinates global gene expression with the nutritional quality of a cell's environment. As a model system for growth rate-dependent regulation of nonribosomal genes, we have been investigating the Escherichia coli gnd gene, which encodes 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44), an enzyme of the pentose phosphate pathway of central carbon metabolism. In E. coli K-12, the specific activity of 6PGD increases about threefold over the fourfold range of growth rates obtained with cells growing in minimal media with various carbon sources, e.g., acetate, in which the doubling time is about 4 h, and glucose, in which the doubling time is about 1 h (25). Antibody neutralization experiments demonstrated that this increase is due to an increase in the amount of 6PGD relative to total protein rather than to an increase in the activity of 6PGD molecules (25). Moreover, since reducing the growth rate by limiting the rate of glucose uptake decreased the amount of 6PGD, gnd gene expression is regulated by the cellular growth rate, not by the specific carbon source (25).Much of the information regarding the mechanism of growth rate ...

RNAs, such as mRNA, rRNA and tRNA, are essential macromolecules for cell survival and maintenance. Any perturbation of these molecules, such as by degradation or mutation, can be toxic to cells and may occasionally induce cell death. Therefore, cells have mechanisms known as quality control systems to eliminate abnormal RNAs. Although tRNA is a stable molecule, the anticodon loop is quite susceptible to tRNA-targeting RNases such as colicin E5 and colicin D. However, the mechanism underlying cellular reaction to tRNA cleavage remains unclear. It had long been believed that tRNA cleavage by colicins E5 and D promptly induces cell death because colony formation of the sensitive cells is severely reduced; this indicates that cells do not resist the tRNA cleavage. Here, we show that Escherichia coli cells enter a bacteriostatic state against the tRNA cleavage of colicins D and E5. The bacteriostasis requires small protein B (SmpB) and transfer-messenger RNA (tmRNA), which are known to mediate trans-translation. Furthermore, another type of colicin, colicin E3 cleaving rRNA, immediately reduces the viability of sensitive cells. Moreover, nascent peptide degradation has an additive effect on bacteriostasis. Considering the recent observation that tRNA cleavage may be used as a means of cell-to-cell communication, tRNA cleavage could be used by bacteria not only to dominate other bacteria living in the same niche, but also to regulate growth of their own or other cells.

Colicins are protein toxins produced by and toxic to Escherichia coli strains. Colicin D consists of an N-terminal domain (NTD), central domain (CD) and C-terminal RNase domain (CRD). The cognate immunity protein, ImmD, is co-synthesized in producer cells to block the toxic tRNase activity of the CRD. Previous studies have reported the crystal structure of CRD/ImmD complex. Colicin D hijacks the surface receptor FepA and the energy transducer TonB system using the NTD for translocation across the outer membrane of the target cells. The CD is required for endoproteolytic processing and the translocation of CRD across the inner membrane, and the membrane-associated protease FtsH and the signal peptidase LepB are exploited in this process. Although several regions of the CD have been identified in interactions with the hijacked inner membrane system or immunity protein, the structural basis of the CD is unknown. In this study, we determined the crystal structure of colicin D, containing both the CD and CRD. The full-length colicin D/ImmD heterodimer structure was built by superimposing the CD-CRD structure with the previously determined partial structures. The overall translocation process of colicin D, including the interaction between CD and LepB, is discussed.