Still looking for the magic spot: the crystallographically defined binding site for ppGpp on RNA polymerase is unlikely to be responsible for rRNA transcription regulation

Abstract

Identification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high-resolution X-ray structure defined a ppGpp binding site on Thermus thermophilus RNAP. We report here effects of ppGpp on 10 mutant Escherichia coli RNAPs with substitutions for the analogous residues within 3-4 A of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of omega, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli ribosomal RNA transcription initiation and highlight the importance of inclusion of omega in bacterial RNAP preparations.

Figure 1

ppGpp binding site in T. thermophilus RNAP. (A) The T. thermophilus ppGpp-RNAP x-ray structure (ref.; PDB coordinates 1SMY) is displayed using PyMol (DeLano Scientific). Subunits are colored as follows: ω, dark purple; α^I, yellow; α^II, green; β, cyan; β′, pink; σ, orange. ppGpp is in yellow spacefill. (B) T. thermophilus RNAP amino acids in close proximity to ppGpp (E. coli RNAP residue numbering). ppGpp is in yellow stick form. Mg²⁺ ions predicted to be coordinated by the proximal and distal (with respect to the active site) ppGpp diphosphates are shown as white spheres. Residues predicted to contact the guanine base of ppGpp (β′ N458, β′ E925, and β′Q929) are in red spacefill, to contact the distal phosphates (β′ K598 and β′ Q504) are in green, to contact the proximal phosphates (β′ R731 and β R1106) are in dark blue, and to contact a ppGpp-coordinated Mg²⁺ (β E813) is in magenta. Substitutions were made for each of these residues and for β E814 and β′ K599 as well (see text), but the T. thermophilus residues corresponding to these amino acids are not pictured because they do not contact ppGpp in the cocrystal. T. thermophilus residues corresponding to the E. coli amino acids in the figure are in parentheses:β′ N458 (N737), β′ Q504 (R783), β′ K598 (K908), β′ R731 (R1029), β′ E925 (E1231), β′ Q929 (Q1235), β R1106 (R879), and β E813 (E685).

Figure 2

Effects of RNAP substitutions on transcription inhibition by ppGpp. (A) Transcription inhibition at saturating ppGpp concentration (400 μM). Multi-round transcription from the rrnB P1 and RNA-I promoters on plasmid pRLG6798 was performed as described (see Materials and Methods). Lanes ± ppGpp are from the same gel in the same experiment. WT = wild-type E. coli RNAP. (B) Transcription was as described in (A) but with 0 to 400 μM ppGpp with wild-type and β′ E925A RNAP. (C) Determination of IC₅₀ for ppGpp and mutant RNAPs. Transcription from rrnB P1 in the experiment shown in (B) was normalized to transcription from RNA-I at each ppGpp concentration (to correct for errors in gel loading) and expressed as a fraction of transcription without ppGpp. The plot allows calculation of the maximal extent of inhibition by ppGpp and the IC₅₀ (ppGpp concentration at which inhibition is half-maximal). Plots for other transcriptionally-active mutants are in Supplementary Fig. 1, and the data are compiled in Table 1.

Figure 3

Effects of ppGpp on promoter complex lifetime of wild-type and β′ N458S mutant RNAP. Fraction of lacUV5 complexes remaining as a function of time after heparin addition at different ppGpp concentrations (see Materials and Methods). Semilog plots for representative experiments: (A) wild-type RNAP, (B) β′ N458S RNAP. (C) Half-lives at each ppGpp concentration. Comparisons of the relative complex half-lives for wild-type RNAP and the other mutant RNAPs are shown in Supplementary Fig. 2, and the data are compiled in Table 2.

Figure 4

Inhibition of mutant RNAPs by ppGpp in the presence of DksA. Single-round transcription was performed as described (see Materials and Methods) with neither ppGpp nor DksA (first lane in each panel), with ppGpp alone (100 μM; second lane in each panel), with DksA alone (third lane in each panel; for concentrations see Materials and Methods), or with both together (fourth lane in each panel). WT = wild-type RNAP. β′ N458S, β′ E925A, and β′ Q929A RNAPs (not shown) exhibited transcription elongation defects, resulting in some incomplete extension products under these conditions (see Materials and Methods).

Figure 5

Tests of competition between ppGpp and the iNTP. (A) ppGpp does not change the effect of the concentration of the first NTP on transcription from rrnB P1. Multi-round transcription from plasmid pRLG6798 containing rrnB P1 was performed ± 1 mM ppGpp at increasing concentrations of ATP (the iNTP) and plotted relative to transcription at 125 μM ATP. (B) iNTP concentration does not affect relative inhibition by ppGpp. Representative gel showing multi-round transcription from rrnB P1 at increasing concentrations of ppGpp with 125 μM or 1500 μM ATP. (C) Transcription from (B) at each ppGpp concentration is plotted as a fraction of transcription without ppGpp.

Figure 6

Effect of ppGpp on transcription initiation by T. thermophilus RNAP. (A) ppGpp (1 mM) does not inhibit transcription by T. thermophilus RNAP. Multi-round transcription from pRLG6770 was measured in 170 mM NaCl transcription buffer at 65°C on a supercoiled template carrying the T. thermophilus 16S rRNA promoter and vector-derived RNA-I promoter (see Materials and Methods). (B) ppGpp (1 mM) does not reduce the lifetime of a promoter complex containing T. thermophilus RNAP. Representative plots show the fraction of heparin-resistant complexes containing T. thermophilus RNAP and the RNA-I promoter on a supercoiled plasmid as a function of time after heparin addition (55°C in 100 mM KCl transcription buffer; see Materials and Methods). The mean ratio of the observed half-lives with/without ppGpp was 0.84 ± 0.17 (3 experiments).