The Feedback Response of Escherichia coli rRNA Synthesis Is Not Identical to the Mechanism of Growth Rate-Dependent Control

Abstract

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...

Changes inEscherichia colirRNA Promoter Activity Correlate with Changes in Initiating Nucleoside Triphosphate and Guanosine 5′ Diphosphate 3′-Diphosphate Concentrations after Induction of Feedback Control of Ribosome Synthesis

“…Therefore, we favor the explanation that there may be additional mediators of feedback control of rRNA synthesis in E. coli. This conclusion is in agreement with that reached by Condon et al and Voulgaris et al, who reported that the ppGpp concentration did not decrease (9) and the ATP concentration did not increase (43) in strains with a reduced number of rRNA operons. Furthermore, these investigators noted that the activities of certain rrn P1 promoter variants that were relatively insensitive to the iNTP concentration were still subject to feedback control in strains with a reduced rrn gene dose.…”

“…Artificial manipulation of ribosome synthesis rates was used previously to demonstrate that a feedback mechanism(s) plays a role in the control of rRNA transcription. For example, when the rRNA gene dose was increased (21) or decreased (9,43), when rRNA was overexpressed from inducible promoters (18), when the fis gene was deleted or the rpoA gene was mutated (4,33,34), or when the rRNA antitermination system was disrupted (39), corresponding changes in rRNA core promoter activities always restored overall rRNA synthesis rates-and therefore ribosome synthesis rates-to the level appropriate for the nutrient condition. Thus, feedback mechanisms balance rRNA promoter activities with the need for protein synthesis.…”

Regulation of rRNA Transcription Correlates with Nucleoside Triphosphate Sensing

“…We also emphasize that the changes in rrnB P1 promoter activity associated with changes in growth rate or loss of the fis gene could be mediated only in part by variations in NTPs, and quantitative discrepancies in the correlations between the in vitro NTP-sensing behavior of the promoters and their in vivo regulatory responses could reflect the participation of other components in the system. Squires and coworkers have suggested that feedback elicited by an increased or decreased rRNA gene dose might be mediated by a mechanism distinct from that responsible for growth rate-dependent regulation and NTP sensing (60). They studied four rrnB P1 variants from our collection (T-33A, Ains-22, C-1T/C-15G, and C-1T) (17) and found an incomplete correlation between the cis-acting sequences required for growth rate-dependent regulation and those required for a response to gene dose changes.…”

Control of rRNA Synthesis in Escherichia coli : a Systems Biology Approach

“…The relationship has been restated with the reference per amount of protein, e.g., "the synthesis of rRNA per unit amount of protein increases with the square of the growth rate and this phenomenon is called growth rate-dependent control of rRNA synthesis" (135). This statement derives from equation 2 above as follows.…”

“…Although the original feedback models addressed the growth rate-dependent control of rRNA synthesis, it was later reported that "the feedback response of E. coli rRNA synthesis is not identical to the mechanism of growth rate-dependent control" (135). In that work feedback response and growth rate-dependent control were defined by the changes in LacZ enzyme expression from rrn P1 resulting from changes in either rrn gene dosage or growth medium, respectively (see the section Current Status of the Field, below).…”