Dissection of the Mechanism for the Stringent Factor RelA Thomas M Wendrich, Gregor Blaha, Daniel N Wilson, Mohamed A Marahiel, Knud H Nierhaus  Molecular Cell  Volume 10, Issue 4, Pages 779-788 (October 2002) DOI: 10.1016/S1097-2765(02)00656-1

Figure 1 Determination of (p)ppGpp Synthesis (A) HPLC separation of nucleotides on a PEI column after (p)ppGpp synthesis. For details and peak assignment, see Experimental Procedures. The nucleoside mono-, di-, and triphosphates elute pairwise (first A, then G). Peak integration revealed that ATP decrements and AMP increments are directly correlated to (p)ppGpp synthesis (±3%), allowing a precise quantification of turnover rates. (B) Representative PEI thin-layer chromatography showing increase in pppGpp synthesis (arrowed) with increasing concentration of ribosomes relative to a constant amount of RelA (1.5 pmol) after a 20 min incubation. The corresponding decrease in ATP can also be seen (also arrowed). Lanes 1–7 represent ribosome:RelA ratios of 0.03, 0.1, 0.3, 1, 3, 10, and 30, respectively. (C) Saturation curve showing (p)ppGpp production when increasing concentrations of 70S ribosomes are titrated against a constant amount of RelA (1.5 pmol). The rate of (p)ppGpp synthesis is given as pmol (p)ppGpp·(pmol RelA·min)−1. The error bars indicate the deviation from the mean. Molecular Cell 2002 10, 779-788DOI: (10.1016/S1097-2765(02)00656-1)

Figure 2 tRNA and Tetracycline Effects on (p)ppGpp Synthesis (A) (p)ppGpp synthesis at various molar ratios of tRNAPhe:70S ribosomes in the presence of poly(U) and RelA. (B) Inhibition of (p)ppGpp synthesis by tetracycline; tRNAPhe was added to poly(U)-programmed 70S ribosomes in a molar ratio of 3:1. The rate of (p)ppGpp synthesis is given as pmol (p)ppGpp·(pmol RelA·min)−1. Molecular Cell 2002 10, 779-788DOI: (10.1016/S1097-2765(02)00656-1)

Figure 3 (p)ppGpp Synthesis Rates of 70S Ribosomes and Ribosomal Subunits (A) Comparison of the (p)ppGpp synthesis rates for complexes of 30S (gray bars) and 50S subunits (white bars) or 70S ribosomes (black bars) in various combinations with RelA, tRNAPhe, and poly(U). (B) (p)ppGpp synthesis rates for L11 minus 70S ribosomes (−L11) in complex with RelA, poly(U), tRNAPhe, and with the addition of purified ribosomal protein L11 (+L11). Values were normalized to pmol (p)ppGpp·(pmolRelA·min)−1. Molecular Cell 2002 10, 779-788DOI: (10.1016/S1097-2765(02)00656-1)

Figure 4 Monitoring tRNA Occupancy at the A and P Sites of the Ribosome in the Presence of MF-mRNA (A) Occupancy of P site alone (bar 1), with addition of GTP/ATP (bar 2) or GTP/ATP and RelA (bar 3) are indicated by white bars. Black bars indicate chasing of f[35S]Met-tRNAfMet with deacylated tRNAfMet added in a two-molar excess over ribosomes, in the presence of GTP/ATP (bar 4) or GTP/ATP with RelA (bar 5). (B) Occupancy of the A site with deacylated [32P]tRNAPhe in the preformed complex with fMet-tRNA at the P site (bar 1 in [A]). Additions of ATP/GTP, or RelA and chasing tRNA are as described in (A), except that chasing was performed with cold deacylated tRNAPhe (black bars). Only conditions in (B) produced strong (p)ppGpp synthesis (>1600 pmol (p)ppGpp·(pmol RelA·min)−1) and are indicated by an asterix (*). All experiments were reproduced in triplicate and have a standard error of up to 5%. Molecular Cell 2002 10, 779-788DOI: (10.1016/S1097-2765(02)00656-1)

Figure 5 Binding of RelA to 70S Ribosomes (A) Elution profile from a Sephacryl S300 spun column of RelA bound to 70S ribosomes in the presence and absence of poly(U) (squares and circles, respectively). Fractions were analyzed by scintillation (filled symbols, left-hand axis) and spectroscopy at A260 nm (open symbols, right-hand axis). (B) SDS-PAGE of RelA and ribosomes. Left side, defined additions of RelA and ribosomes per lane. Right side, quantification of eluted RelA:70S ribosome fractions by Western immunoblotting (from fraction 1 of a spun column analysis as in [A]). RelA was detected with anti-His5 by luminol reaction and L2 with anti-L2 (sheep) by alkaline phosphatase reaction (anti-sheep-AP conjugate). Molecular Cell 2002 10, 779-788DOI: (10.1016/S1097-2765(02)00656-1)

Figure 6 RelA Binding to Ribosomal Complexes (A) Binding to 70S ribosomes, and where indicated, in the presence of poly(U) and tRNAPhe. (B) RelA binding to 70S ribosomes programmed with various mRNAs: poly(U), MS2 mRNA, or MF-mRNA. (C) RelA binding to poly(U)-programmed 70S ribosomes in the presence of tRNAPhe, and where indicated, supplemented with ATP, GTP, or γS-ATP at 1 mM. Left (black bars), 70S containing L11; right (white bars), 70S lacking L11. The first fraction from a spun column treatment as in Figure 5A was used for ratio determination (deviation ±5%) as described in Experimental Procedures. EF-Tu indicates delivery of Phe-tRNAPhe to the A site in the form of the ternary complex Phe-tRNA·EF-Tu·GTP. We note that a binding value of 0.66/70S of the ternary complex was not affected by the addition of RelA. Molecular Cell 2002 10, 779-788DOI: (10.1016/S1097-2765(02)00656-1)

Figure 7 Model for Mechanism of RelA-Mediated (p)ppGpp Synthesis (a) Amino acid starvation generates large pools of deacylated tRNAs, which bind to the ribosomal A site with low affinity and block the ribosome. (b) RelA detects a blocked ribosome with a 3′ extension of the mRNA. (c) RelA mediates the conversion of ATP and GTP(GDP) to AMP and (p)ppGpp in the presence of a deacylated tRNA at the A site. Release of RelA but not the A site-bound deacylated tRNA occurs simultaneously with RelA-mediated (p)ppGpp synthesis. (d) RelA “hops” to the next blocked ribosome, and the synthesis of (p)ppGpp is repeated. High levels of (p)ppGpp activate the stringent response. (e) Aminoacylated tRNAs are replenished following post-stress conditions. The higher affinity of an aminoacylated tRNA over deacylated tRNAs for the A site enables displacement of the deacylated tRNAs, which rescues blocked ribosomes and reactivates translation. Note that binding of an aminoacylated tRNA at the A site also results in concomitant release of the E site bound tRNA (reviewed by Nierhaus, 1990); thus two deacylated tRNAs are released. Molecular Cell 2002 10, 779-788DOI: (10.1016/S1097-2765(02)00656-1)