1. Control of rRNA Expression by Small Molecules Is Dynamic and Nonredundant 
Heath D. Murray, David A. Schneider, Richard L. Gourse 
Molecular Cell 
Volume 12, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1
Regulation of rRNA Promoters during Outgrowth from Stationary Phase
(A) Sequences of promoter regions. −10 and −35 hexamers (the core promoter) are indicated in bold, and initiating nucleotides are capitalized. rrnB P1 is referred to here as rrnB P1(+1A) because it initiates with ATP. rrnB P1(+1G) is identical to rrnB P1(+1A) except for an A to G substitution at the transcription start site, leading to initiation with GTP rather than ATP (Schneider et al., 2002). An rrnB P1 derivative, rrnB P1(dis), which contains a 3 bp substitution (underlined) in the region between the transcription start site and the −10 hexamer, was shown previously to be unresponsive to changes in growth rate or amino acid starvation (Barker et al., 2001) and therefore was used as a control promoter. The lacUV5 promoter served as an additional control not regulated by the system's controlling rRNA promoters.
(B) Representative primer extension results for promoter activity determinations. In order to visualize the amount of transcription at time zero, 2-fold more sample was loaded on the gel than at the other time points.
(C) Representative chromatogram for determination of NTP and ppGpp concentrations. The region of the chromatogram containing ppGpp was exposed longer than that containing ATP and GTP. In order to visualize the nucleotides at time zero, 10-fold more sample was loaded than for the other time points.
(D) Relative promoter activities during outgrowth. The activity of each promoter has been normalized to a value of 1.0 at time zero, after correction for the amount of sample loaded on the gel. The amounts of rrn P1 transcripts at time zero were also evaluated with greater precision by electrophoresis on higher resolution gels with longer phosphorimaging (data not shown), and it was found that the relative increase in rrn P1 promoter activity during outgrowth in these experiments was at least as great as that illustrated in (D).
(E) Nucleotide concentrations during outgrowth (arbitrary units, a.u.). The concentrations are not normalized in this panel, allowing comparison of the absolute levels of ATP, GTP, and ppGpp.
(F and G) Relative promoter activities (F) and relative NTP and ppGpp concentrations (G) during outgrowth in a purK strain supplemented with adenine (see Experimental Procedures and Table 1). Concentrations were normalized to their values at time zero.
(H and I) Relative promoter activities (H) and relative NTP and ppGpp concentrations (I) during outgrowth in a purK strain supplemented with guanine. Averages and standard deviations of promoter activities, ppGpp, and NTP concentrations were from at least three independent experiments.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Regulation of rRNA Promoter Activity during Upshifts and Downshifts
(A and B) Relative promoter activities (A) and relative ATP and ppGpp concentrations (B) following upshift of a wild-type strain after addition of amino acids to minimal glycerol medium.
(C) Relative promoter activities in wild-type versus ΔrelAΔspoT strains following upshift by addition of brain heart infusion medium to glycerol-defined medium containing casamino acids.
(D and E) Relative promoter activities (D) and relative ATP and ppGpp concentrations (E) in a wild-type strain during downshift by addition of α-methyl glucoside.
(F) Relative promoter activities after addition of α-methyl glucoside to wild-type versus ΔrelA strains (ΔrelAΔspoT strains were not used since they are unable to grow without amino acids; Xiao et al., 1991). All promoter activities and nucleotide concentrations were normalized to their respective values at time zero.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Regulation of rRNA Promoter Activity during Entry into and Maintenance of Stationary Phase
(A and B) Relative promoter activities (A) and relative ATP and ppGpp concentrations (B) in wild-type strains. Promoter activities and ATP measurements are illustrated starting at OD600 ∼0.1 (200 min). ATP and ppGpp concentrations were both normalized to the ATP concentration at 200 min, allowing comparison of their absolute concentrations. Cell densities were too low for detection of ppGpp until ∼325 min. The arrow at ∼400 min (OD600 ∼1.0) indicates when rrnB P1 promoter activity began to decrease, concurrent with an increase in the ppGpp concentration and a decrease in the ATP concentration.
(C) Relative promoter activities in wild-type VH1000 and ΔrelAΔspoT strains.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Homeostatic Regulation of rRNA Promoters by Small Molecules
(A) Summary of stages in growth when changing concentrations of ppGpp and/or iNTP regulate transcription from rrn P1 promoters. Yellow, outgrowth; green, exponential phase. The dotted arrows originating from the exponential portion of the growth curve are used to indicate an upshift to a medium resulting in an increase in growth rate (decreased ppGpp) or a downshift to a medium resulting in a decrease in growth rate (increased ppGpp). Blue, stationary phase.
(B) Schematic diagram illustrating two feedback circuits proposed to control rRNA expression. In one, rrn P1 promoters sense overproduction of ribosomes by transient depletion of iNTPs resulting from the process of translation. In the other, rrn P1 promoters sense overinvestment in translational capacity by transient production of ppGpp resulting from uncharged tRNAs in the ribosomal A site. The kinetic scheme illustrates the steps in the rrn P1 transcription initiation mechanism. RPc, closed complex. RPo, open complex. RPNTP, open complex containing iNTP. ppGpp stimulates conversion of RPo to RPc (Barker et al., 2001). The iNTP stabilizes RPo (Gaal et al., 1997). The cloverleafs and dashed line symbolize tRNAs and the mRNA, respectively. The gray ovals symbolize amino acids in the peptide chain attached to the P site tRNA. RelA is the major ppGpp synthetase (Cashel et al., 1996).
Molecular Cell  , DOI: ( /S (03) )