Effect of polyamines on the inhibition of peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action

Abstract

Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3'-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg2+, 100 mM NH4+, 100 microM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA.poly(U).70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes.

Figure 1

First-order time plots for the AcPhe-puromycin synthesis in the presence or absence of chloramphenicol. Complex C reacted at 25°C in the presence of 6 mM Mg²⁺ and 100 mM NH₄⁺, with (open circles) 400 µM puromycin or with a solution containing 400 µM puromycin and chloramphenicol at (filled circles) 3 µM, (open squares) 6 µM, (filled squares) 12 µM and (diamonds) 18 µM. The deviation from linearity observed in the presence of chloramphenicol reveals a delay on the onset of inhibition.

Figure 2

Double-reciprocal plots for the AcPhe-puromycin synthesis in the presence or absence of chloramphenicol. The data presented in panels A and B were collected from the early (t < 15 s) and the late phases (t > 1 min) of logarithmic time plots, respectively, such as those shown in Figure 1. The reaction was carried out at 6 mM Mg²⁺ and 100 mM NH₄⁺, in the absence (open circles) or in the presence of chloramphenicol at (filled circles) 3 µM, (open squares) 6 µM, (filled squares) 12 µM and (diamonds) 18 µM. The inhibition at both phases of the reaction, early and late, is of the competitive type.

Figure 3

Slope replots (slopes of double-reciprocal plots versus chloramphenicol concentration). The data were obtained from the double-reciprocal plots of (open circles) Figure 2A and (filled circles) 2B. The linearity of slope replots confirms the competitive character of inhibition and indicates that only one inhibitor binding site is implicated in both phases of the reaction.

Figure 4

Variation of the apparent equilibration rate constant, k′, as a function of chloramphenicol concentration. The reaction was carried out at 6 mM Mg²⁺ and 100 mM NH₄⁺ in the presence of puromycin at (filled circles) 2 mM, (open circles) 400 µM, (triangles) 200 µM and (squares) 100 µM. The k′ value at each concentration of puromycin and chloramphenicol was estimated from the intersection point of the two linear parts of the corresponding progress curve (for example, see lower line in Fig. 1). The hyperbolic character of plots reveals that chloramphenicol inhibits the puromycin reaction by a two-step mechanism.

Figure 5

Analysis of ABA-spermine cross-links in 23S rRNA by RNase H digestion. 23S rRNA from complex C photolabeled with 100 µM ABA-[¹⁴C]spermine, was incubated with cDNA probes, and then digested with RNase H. The products of digestion were resolved on 5% polyacrylamide/ 7 M urea gels and visualized by autoradiography. Lanes 1 and 2, undigested samples from complex C irradiated in the absence and presence of excess spermine, respectively. Lanes 3 to 6, samples hybridized (lane 3) with cDNAs 1962–1973 and 2159–2169, (lane 4) with cDNAs 2159–2169 and 2350–2360, (lane 5) with cDNAs 2350–2360 and 2568–2578, (lane 6) with cDNAs 2568–2578 and 2781–2791, and then digested with RNase H. Numbers indicated to the left of the gel correspond to the sizes (nt) of RNA markers. The region encompassing domain V of 23S rRNA, appears to be susceptible to ABA-spermine cross-linking.

Figure 6

Gel electrophoresis of products resulting from ABA-spermine photoincorporation into complex C, as monitored by primer extension analysis. The primers for reverse transcription were complementary to 23S rRNA positions (A) 2560–2576, and (B) 2697–2681. Positions at 10-nt intervals, read from sequencing products (lanes U, A, G and C), are shown in the left margin of each panel. Lane 1, untreated complex C; lane 2, complex C photolabeled with 100 µM ABA-spermine; lane 3, complex C photolabeled with 100 µM ABA-spermine in the presence of a 250-fold excess of spermine; lane 4, complex C modified by DMS; lane 5, complex C photolabeled with 100 µM ABA-spermine, and then modified by DMS. The stops of reverse transcriptase reaction are indicated in the right margin of each panel by arrows. SPM, spermine; DMS, dimethyl sulfate. Nucleosides in the central loop of domain V and in helices projecting from this loop (H89, H90 and H93) constitute preferred cross-linking sites for ABA-spermine. Upon ABA-spermine cross-linking, these regions undergo alterations in their reactivity towards DMS.

Figure 7

Summary of ABA-spermine cross-linking sites in the central region of domain V of E.coli 23S rRNA. Cross-linking sites in a secondary structure model (cited at http://www.ma.icmb.utexas.edu) are marked with green arrows. Sites sensitive to divalent metal ion-catalyzed hydrolysis (38) are indicated by red arrows. Sites implicated in chloramphenicol binding (–19) are shown by blue letters. ABA-spermine cross-linking sites and metal binding sites do not always coincide. Although the region encompassing nucleosides 2057–2062 constitutes a primary target for chloramphenicol binding, no ABA-spermine cross-linking is detected within this region.

Scheme 1.