Context-Specific Action of Ribosomal Antibiotics

Abstract

The ribosome is a major antibiotic target. Many types of inhibitors can stop cells from growing by binding at functional centers of the ribosome and interfering with its ability to synthesize proteins. These antibiotics were usually viewed as general protein synthesis inhibitors, which indiscriminately stop translation at every codon of every mRNA, preventing the ribosome from making any protein. However, at each step of the translation cycle, the ribosome interacts with multiple ligands (mRNAs, tRNA substrates, translation factors, etc.), and as a result, the properties of the translation complex vary from codon to codon and from gene to gene. Therefore, rather than being indiscriminate inhibitors, many ribosomal antibiotics impact protein synthesis in a context-specific manner. This review presents a snapshot of the growing body of evidence that some, and possibly most, ribosome-targeting antibiotics manifest site specificity of action, which is modulated by the nature of the nascent protein, the mRNA, or the tRNAs.

Keywords: antibiotic; chloramphenicol; erythromycin; kasugamycin; ketolides; linezolid; macrolides; nascent peptide exit tunnel; pactamycin; peptidyl transferase center; protein synthesis; resistance; ribosome; translation.

Figure 1.

An overview of protein synthesis and the steps inhibited by antibiotics with context-specific mode of action. At the initiation step, the ribosome, assisted by initiation factors (IF1-3), locates the start codon in mRNA and binds the initiator tRNA in the P site. Kasugamycin selectively interferes with initiation of translation of some mRNAs depending on the structure of their ribosome binding site. During translation elongation, EF-Tu-delivered aminoacyl-tRNAs are selected and then accommodated in the A site. As the peptide bond is formed in the peptidyl transferase center (PTC), the growing nascent protein is transferred to the A site tRNA. The antibiotic-mediated direct (chloramphenicol and linezolid) or allosteric (macrolides) inhibition of peptide bond formation depends on the structure of the nascent protein. Subsequent translocation of the ribosome is promoted by EF-G. Inhibition of translocation by pactamycin may depend on the nature of the A site substrate. During translation elongation, the growing nascent protein is threaded through the nascent peptide exit tunnel (NPET). When the ribosome encounters a stop codon, it enters the termination phase, during which the completed protein is released with the help of termination factors (RF1 or RF2, and RF3). Finally, at the recycling phase, the combined action of ribosome recycling factor (RRF) and EF-G splits the ribosome into its subunits.

Figure 2.

Methodologies for studying the context specific action of ribosomal antibiotics. (a) Ribo-Seq, also known as ribosome profiling, involves the isolation, deep-sequencing and mapping to the genome of ribosomal footprints, the mRNA fragments associated with the translating ribosomes in the living cell. Analysis of antibiotic-induced changes in the ribosomal footprints pattern at the codon or gene level reveals that the action of several antibiotic families is context specific. (b) In vitro toeprinting analysis allows for mapping the position of an arrested ribosome in mRNA with codon precision. Top: extension of a DNA primer (black arrow) by reverse transcriptase (not shown) is interrupted by the presence of a stalled ribosome. The 1^st nucleotide of the codon occupying the P site and the 3’-terminal end of the truncated-cDNA are separated by a distance of 15-16 nucleotides. Bottom: the cDNA products, along with sequencing reactions, are resolved in a sequencing gel. The position of the stalled ribosome is inferred from the migration of the toeprint band. Antibiotic-dependent appearance of toeprint bands reveals the specific codons where translation is arrested.

Figure 3.

The context specific action of PTC inhibitors chloramphenicol (CHL) and linezolid (LZD). (a) The chemical structures of CHL and LZD. (b) The binding site of CHL and LZD overlaps with that of the aminoacyl moiety of the A site aa-tRNA. (c) RiboSeq analysis shows that CHL (and LZD) causes redistribution of ribosomes on mRNA (51). In the presence of either one of the inhibitors, ribosomes are preferentially arrested at the codons that immediately follow Ala codons. (d) Cartoon representation of the preferred target of CHL (or LZD), a ribosome carrying a nascent peptide whose penultimate residue is an alanine.

Figure 4.

Macrolide antibiotics halt translation of proteins carrying specific sequence motifs. (a) The chemical structures of the prototype macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). (b) Macrolides and ketolides bind at the NPET and partially obstruct the passage of the newly made protein. A and P site tRNAs are also shown. (c) Ribo-Seq data demonstrate that TEL (as well as other macrolides) causes translation arrest at discrete and specific sites along mRNAs (41). (d) Two-dimensional electrophoretic analysis of the proteins synthesized in E. coli cells exposed to saturating concentrations of ERY, TEL, or the natural ketolide pikromycin (PKM). (e) The different paths of the ErmBL and ErmCL nascent peptides in the NPET partially obstructed by the presence of an ERY molecule (4).

Figure 5.

Kasugamycin (KSG) action depends on the structure of mRNA while the activity of pactamycin (PAC) relies on the nature of A site tRNA. (a) Chemical structures of KSG and PAC. (b) KSG and PAC bind to the small ribosomal subunit and alter the path of the mRNA in the E site. (c) KSG efficiently inhibits translation of leadered mRNAs containing SD sequence, while translation of leaderless transcripts continues in the presence of the antibiotic. (c) Toeprinting analysis gel illustrating that PAC inhibits in vitro translation of the at specific codons of ermBL mRNA (62).

Figure 6.

A highly specific small molecule inhibitor of the human ribosome whose activity depends on the sequence of the nascent protein. (a) The chemical structure of compound PF-06446846. (b) Ribosome profiling shows that PF-06446846 specifically arrest translation at codon 34 of the human gene PCSK9 (47). (c) The amino acid sequences of the nascent peptides at the sites of translation arrest induced by PF-06446846 and the incoming A site amino acid (47). The codon number within the corresponding gene at which arrest occurs is indicated by the number to the right of the sequence.

Figure 7.

Activation of antibiotics resistance genes relies on the context-specific action of ribosomal-targeting antibiotics. (a) Programmed translation arrest at the leader ORF mediates the change in mRNA conformation necessary to activate expression of the resistance gene. (b) Many of the regulatory peptides responsible for activating macrolide resistance genes contain the sequence motif +x+, a prevalent arrest motif for macrolide antibiotics.