Carbon starvation and growth rate-dependent regulation of the Escherichia coli ribosomal RNA promoters: differential control of dual promoters.

Abstract

We studied the effects of carbon starvation and of varying the growth rate on the activity of each of the two tandem ribosomal RNA promoters from the rrnA operon of Escherichia coli. The cellular abundance of plasmid-encoded transcripts arising at promoters P1 and P2 and terminating at the ribosomal RNA terminator in promoter-terminator fusions, together with transcript turnover rates, was used to estimate promoter activities. The rate of synthesis of the P1-promoted transcript was found to increase exponentially with growth rate and predominate at fast growth rates. The activity of the downstream promoter (P2) changed only slightly at different growth rates. Upon carbon starvation, little or no activity of the upstream promoter was detectable, while P2 activity persisted. We interpret this to mean that the dual promoters are differentially regulated so as to have separate adaptive and maintenance functions. This model simplifies most features of rRNA regulation known in E coli. In vitro studies have resulted in proposals of a variety of mechanisms by which rrn transcription regulation could occur (1, 2). In vivo studies have localized regulatory determinants with increasing precision. Fusions of rrn promoter regions with portions of the galactose operon have shown that the target for both stringent and growth rate control is within the promoter region (9, 10). Probably all of the E. coli rrn operons have dual promoters, although one remains that has not had its sequence determined. We have been able to measure the activity of each of the two rrinA promoters in vivo by using plasmids containing rrn promoter-terminator fusions (11,12). With rapidly growing cells, the upstream P1 promoter was found to be about three times more active than the downstream P2 promoter. During the stringent RNA control response, P1 was about 90% inhibited in relA+ but not in relA strains, indicating stringent control of P1 activity. In contrast, P2 was judged to be only moderately (50%) inhibited in both relA+ and relA hosts and, therefore, was not under stringent control. Furthermore, stringent regulation of P1 activity persisted even when the P2 promoter and the downstream regions extending to the mature 16S RNA gene were deleted (11,13).Here we describe measurements of the relative activities of the rrnA P1 and P2 promoters as a function of growth rate variation and during glucose starvation. We have found that the P1 promoter is strongly dependent on growth rate and can be progressively inactivated as growth slows to the point where, as during glucose starvation, it is only marginally detectable. The downstream P2 promoter behaves very differently; its activity is only weakly dependent on growth rate and remains quite active during glucose starvation. We suggest that the downstream promoter behaves as a constitutive maintenance promoter whose activity is relatively insensitive to regulation by either the stringent or growth-rate control mechanisms. This behavior can account for the excessive synthesis of rRNA in very slow...

Conserved features of coordinately regulatedE.colipromoters

“…Within this class there are different responses to variation of growth rate. Thus the rate of expression of the rrnA P1 promoter is approximately proportional to the square of the growth rate (19) while those of the leuV and tyrT promoters are directly proportional (20,21). By contrast expression of the rpsE and rpsMR promoters is independent of growth rate, variation in the levels of the cognate mRNA species being achieved possibly by post transcriptional controls (22).…”

Comparison of Bacteroides thetaiotaomicron and Escherichia coli 16S rRNA gene expression signals

“…E. coli rRNA synthesis is regulated by the collective response of the dual promoters to growth rate (Gourse et al, 1996), amino acid starvation (Cashel et al, 1996;Gafny et al, 1994) and rRNA gene dose (Gourse et al, 1996). P1 is considered the stronger of the two promoters since it accounts for most of rRNA transcription at all but the slowest growth rates (Sarmientos & Cashel, 1983). It has been suggested that P2 promoters are inhibited by transcription from P1 (Gafny et al, 1994).…”

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli

“…The magnitude of the signal made in response to the cell's translational capacity varies with the nutritional state of the culture. The identity of the signal is not known, although guanosine tetraphosphate (ppGpp) has been implicated as playing some role because of the virtually perfect inverse correlation between stable RNA synthesis and ppGpp concentration (36).The target of the signal, whatever its identity, has been shown in at least three of the seven rRNA operons to be P1, the more upstream of the two rRNA promoters (rrnB and rrnE [15]; rrnA [37] there is a region called the upstream activation sequence (UAS), which is required for maximal promoter activity (4,15,23).Mutations have been targeted to specific sites in the promoters rrnB P1 and tyrT, and activities resulting from the fusion of the mutant promoters to "reporter" genes have been measured under different growth conditions (15,46). Such experiments have implicated DNA sequences required for growth rate-dependent control in the region between -20 and -50 (15) and in the region just downstream of the -10 hexamer (46).…”

“…The target of the signal, whatever its identity, has been shown in at least three of the seven rRNA operons to be P1, the more upstream of the two rRNA promoters (rrnB and rrnE [15]; rrnA [37]). The DNA sequence determinants responsible for growth rate regulation are limited to positions between -4 and -50 with respect to the transcription start site (15).…”

“…The target of the signal, whatever its identity, has been shown in at least three of the seven rRNA operons to be P1, the more upstream of the two rRNA promoters (rrnB and rrnE [15]; rrnA [37] there is a region called the upstream activation sequence (UAS), which is required for maximal promoter activity (4,15,23).…”