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Antibiotic Blocks Mrna Path on the Ribosome NEWS AND VIEWS Antibiotic blocks mRNA path on the ribosome Alexander Mankin Many translation initiation inhibitors block transfer RNA binding or placement in the ribosome. Structures of kasugamycin bound to the bacterial ribosome now indicate that it instead blocks proper mRNA placement. In 1965, a compound was isolated from a EPA mRNA EPA EPA Streptomyces strain found near the Kasuga a SD SD SD shrine in Nara, Japan. The drug could pre- http://www.nature.com/nsmb Small vent growth of a fungus causing rice blast subunit Subsequent steps of disease and later was found to inhibit bacte- translation fMet-tRNA Large rial growth. It was called kasugamycin. subunit Like many (actually, almost half) of all the known natural antibiotics, kasugamycin inhibits proliferation of bacteria by tamper- Kasugamycin ing with their ability to make new proteins, EPA EPA the ribosome being the major target of such b SD SD x drugs. In essence, the ribosome is an RNA Initiation is blocked machine, and most antibiotics that inhibit Nature Publishing Group Group Nature Publishing translation bind ribosomal RNA and affect 6 rRNA interactions with the ligands of the 200 ribosome: aminoacyl- and peptidyl-tRNAs, EPA EPA © translation factors or the nascent peptide. c A great variety of drugs that target the ribo- some inhibit elongation of translation. In Subsequent contrast, only a small fraction of the known steps of translation protein-synthesis inhibitors affect transla- tion initiation. In many cases, the mechanism of action of such drugs is obscure. During translation initiation, the small ribosomal sub- unit locates a specific segment in mRNA known EPA EPA as the translation-initiation region (Fig. 1a)1. d Translation can proceed With the assistance of translation-initiation factors, the small subunit directs initiator fMet-tRNA to the mRNA start codon. Once initiator tRNA is placed in the small subunit’s peptidyl-tRNA (P) site, the complex is joined by the large ribosomal subunit. Aminoacyl- tRNA is then delivered to the A site by the EF-Tu–GTP ternary complex. Formation of the Figure 1 Inhibition of translation initiation in bacteria by kasugamycin. (a,b) Kasugamycin inhibits first peptide bond and translocation represents translation initiation on mRNA with a 5ʹ leader sequence. a depicts bacterial translation initiation on the transition from the initiation step to the mRNA containing a 5ʹ leader carrying a Shine-Dalgarno sequence (SD). The small ribosomal subunit binds the translation-initiation region of mRNA and (with the help of initiation factors) promotes binding of initiator fMet-tRNA to the start codon positioned in the P site. The subsequent steps of Alexander Mankin is at the Center for translation ensue. Kasugamycin binding (b) overlaps with the last two nucleotides of the E-site codon and the first nucleotide of the P-site codon, likely perturbing mRNA placement in the ribosome and Pharmaceutical Biotechnology, The University preventing efficient binding of initiator tRNA. (c,d) Translation initiation on leaderless mRNA may start of Illinois, 900 S. Ashland Ave., Rm. 3056, with binding of the complete ribosome to the mRNA 5ʹ end (c) and is less kasugamycin sensitive. As Chicago, Illinois 60607, USA. kasugamycin overlaps with only one mRNA nucleotide and initiator tRNA binding is stabilized by its e-mail: [email protected] interaction with the 50S ribosomal subunit, initiation can proceed even in the presence of the drug (d). 858 VOLUME 13 NUMBER 10 OCTOBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY NEWS AND VIEWS elongation step of translation. The translation- that kasugamycin overlaps with the position initiation region in mRNA is composed of two of three nucleotides of mRNA: the last two tRNA anticodon elements: the initiator AUG codon and a Shine- nucleotides of the E-site codon and the first Dalgarno region—a short nucleotide sequence nucleotide of the P-site codon (Figs. 1 and 2). Pct complementary to the 3ʹ end of the small ribo- Therefore, kasugamycin must inhibit bind- Edn somal subunit RNA2. These two elements are ing of the fMet-tRNA to the 30S initiation separated in mRNA by a short, 5- to 9- nucleotide complex only indirectly. This distinguishes spacer. In the context of the initiation complex, kasugamycin from edeine, a chemically differ- this mRNA spacer traverses the vacant exit (E) ent inhibitor of translation initiation, which Ksg2 site, which in the elongating ribosome is tempo- interacts with the same ribosomal site (Fig. 2)8. Ksg1 rarily occupied by deacylated tRNA. Edeine directly clashes with fMet-tRNA in the E site P site Kasugamycin has long been known to inhibit ribosomal P site8,9. Thus, two drugs interacting Figure 2 The binding sites of two kasugamycin binding of initiator fMet-tRNA to the mRNA- with almost the same site in the ribosome have molecules (Ksg1 and Ksg2) on the T. thermophilus programmed small ribosomal subunit during markedly different mechanisms of action. small ribosomal subunit overlap with the sites translation initiation3. The conventional view In principle, kasugamycin might either of action of edeine (Edn) and pactamycin (Pct). of the mode of kasugamycin action came from prevent mRNA binding to the small ribosomal Green, mRNA; orange, anticodon stem-loop of genetic and biochemical studies. RNA foot- subunit or, alternatively, displace mRNA P-site tRNA. Figure courtesy of Daniel Wilson. printing experiments showed that kasugamycin from its normal path. Though some weak protects two nucleotides in the rRNA of the competition between kasugamycin and mRNA Escherichia coli small ribosomal subunit, A794 was observed5, the most probable scenario come its partial destabilization resulting from and G926. Mutations at these positions confer is that the drug repositions mRNA in the the displacement of mRNA by kasugamycin. http://www.nature.com/nsmb resistance to the drug. In the classic footprinting initiation complex, leading eventually to a less Though the two new structures of experiments of Moazed and Noller4, positions efficient interaction of the initiator tRNA with kasugamycin–ribosome complexes are rather A794 and G926 were also found to be pro- the AUG codon. The action of kasugamycin similar, there is one important difference. tected by tRNA bound in the ribosomal P site. clearly depends not only on its clash with the Whereas only one kasugamycin molecule Therefore, it was perfectly reasonable to assume first nucleotide of the mRNA initiator AUG is seen bound to the E. coli 70S ribosomes, that kasugamycin interferes with initiation codon in the P site but also on displacement of two molecules of kasugamycin are clearly of translation by directly hindering binding the mRNA from the E site. Indeed, Schuwirth bound to the T. thermophilus small ribosomal of peptidyl-tRNA. However, two papers that et al.6 show that inhibition of protein synthesis subunit. In the T. thermophilus structure, appear on pages 871 and 879 of this issue show by kasugamycin depends on the identities of the second molecule of kasugamycin binds that this view requires substantial revision. the two mRNA nucleotides that immediately right next to the first one—also on the path Nature Publishing Group Group Nature Publishing Two independent teams used crystallo- precede the AUG codon and that therefore of mRNA through the E site (Fig. 2). The 6 graphy to understand where kasugamycin occupy the E site during translation initia- finding of the second kasugamycin binding 200 binds the ribosome and how it inhibits pro- tion. This result corresponds well to reports site is puzzling. Studies of resistance mutations © tein synthesis5,6. Whereas Schluenzen et al.5 that synthesis of different proteins in the cell clearly show that the first site is the main soaked the drug into crystals of the Thermus is inhibited to different extents by kasuga- site of kasugamycin action: all the known thermophilus small ribosomal subunit, mycin10,11. Thus, the ribosomal E site and its mutations are expected to affect binding of a Schuwirth et al.6 made use of the complete possible interaction with the mRNA spacer kasugamycin molecule to that site. Nevertheless, E. coli ribosome, whose structure had recently connecting the Shine-Dalgarno sequence and the location of the second site is provocative, been reported7. In spite of the differences in the the AUG codon may be important not only in as it almost precisely coincides with the site source and composition of the ribosomes as elongation but also in initiation of translation. of action of another antibiotic, pactamycin14 well as the crystallization conditions, the struc- The overlap of the kasugamycin-binding (Fig. 2). Pactamycin has long been considered tures (solved at 3.35 Å and 3.5 Å in refs. 5 and site with the mRNA segment upstream of an inhibitor of translation initiation, though 6, respectively) yield similar conclusions about the AUG codon sheds light on another pecu- more recent studies have shown that it the position of kasugamycin in the bacterial liarity of the drug’s action. It has long been interferes with translocation9. Interestingly, like ribosome. In its main site, the drug is bound known that kasugamycin inhibits translation kasugamycin, pactamycin may have specific in the cleft between the head and the platform of mRNAs equipped with a Shine-Dalgarno effects on different mRNA sequences in the of the small ribosomal subunit. This placement sequence but is markedly less efficient in 30S subunit E site9. It is difficult to believe that of kasugamycin fits well with the genetic and inhibiting translation of leaderless mRNAs, kasugamycin could have evolved to bind two biochemical evidence, as the drug makes direct which begin with the initiator AUG codon at structurally different ribosomal sites. However, contact with A794 and G926. Furthermore, it is their extreme 5ʹ end. Initiation of translation it is possible that, like the peptidyl-transferase now clear that mutation of either of these two of such mRNAs may proceed through direct center of the ribosome which can accommodate nucleotides directly affects the drug’s binding.
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