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 ...