The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrndeletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrndeletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

When the number of rRNA (rrn) operons in anEscherichia coli cells is increased by adding anrrn operon on a multicopy plasmid, the rate of rRNA expression per operon is reduced to maintain a constant concentration of rRNA in the cell. We have used electron microscopy to examine rRNA transcription in cells containing a multicopy plasmid carryingrrnB. We found that there were fewer RNA polymerase molecules transcribing the rrn genes, as predicted from previous gene dosage studies. Furthermore, RNA polymerase molecules were arranged in irregularly spaced groups along the operon. No apparent pause or transcription termination sites that would account for the irregular spacing of the groups of polymerase molecules were observed. We also found that the overall transcription elongation rate was unchanged when the rrn gene dosage was increased. Our data suggest that when rrn gene dosage is increased, initiation events, or promoter-proximal elongation events, are interrupted at irregular time intervals.

Growth rate-independent rrn P1 promoter mutants were tested for their ability to respond to changes in rrn gene dosage. Most were found to be normal for the feedback response. In addition, cellular levels of the initiating nucleoside triphosphates remained unchanged when the rrn gene dosage was altered. These results suggest that the feedback response cannot be the mechanism for growth rate-dependent control of rRNA synthesis and that the relationship between these two processes may be more complicated than is currently understood.In rapidly growing Escherichia coli cultures, the level of rRNA synthesis controls the protein biosynthetic capacity of the cells (for reviews, see references 5 and 11). The synthesis of rRNA per unit amount of protein increases with the square of the growth rate (14); this phenomenon is called growth rate-dependent control. However, when rrn gene dosage is increased (by the addition of an rrn operon on a multicopy plasmid) or decreased (by deletion of rrn operons from the chromosome), expression from the individual rrn operons changes so that the total rRNA content stays the same (3,4,12,17). This effect is called the feedback response. The rrn P1 promoter is both the site of growth rate-dependent control and the target site of feedback control, suggesting that the feedback response might be the mechanism of growth rate-dependent control (2,6,7,10).A model correlating initiating nucleoside triphosphate (NTP) concentration with growth rate-dependent control has been proposed (9). In vitro, open complexes of the rrn P1 promoters require high concentrations of either GTP (the initiating nucleotide for rrnD) or ATP (the initiating nucleotide for the remaining six rrn operons). Correspondingly, in vivo, NTP concentrations rise in the cell as the growth rate increases. Also, one growth rate-independent P1 mutant loses the ability to respond to changes in the initiating nucleotide. To explain these results, Gaal et al. propose a model in which an increase in nutrient availability (and hence an increase in NTP concentration) leads to increased rRNA synthesis until a level is reached in which the availability of NTPs is in equilibrium with the consumption of ATP and GTP by the protein synthetic apparatus. These authors suggest that this model can also explain the feedback response: a change in the number of ribosomes as the result of altered gene dosage can lead to a change in ATP and GTP consumption, thus affecting the level of rrn transcription.In this study, we have sought to determine if the feedback response and growth rate-dependent control are analogous processes. We have approached this question by asking if promoter elements that are required for growth rate-dependent control are the same as those required for the feedback response. Specifically, we tested a representative sample of rrn P1 promoters, which are mutated so that they are now growth rate independent, for a response to changes in rrn gene dosage. One of the mutations, a single base substitution at Ϫ1 (C-1T; JV2979) does no...