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NEWS AND VIEWS © 2006 Nature Publishing Group http://www.nature.com/nsmb 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 Streptomyces strain found near the Kasuga shrine in Nara, Japan. The drug could prevent growth of a fungus causing rice blast disease and later was found to inhibit bacterial growth. It was called kasugamycin. Like many (actually, almost half) of all the known natural antibiotics, kasugamycin inhibits proliferation of bacteria by tampering with their ability to make new proteins, the ribosome being the major target of such drugs. In essence, the ribosome is an RNA machine, and most antibiotics that inhibit translation bind ribosomal RNA and affect rRNA interactions with the ligands of the ribosome: aminoacyl- and peptidyl-tRNAs, translation factors or the nascent peptide. A great variety of drugs that target the ribosome inhibit elongation of translation. In contrast, only a small fraction of the known protein-synthesis inhibitors affect translation initiation. In many cases, the mechanism of action of such drugs is obscure. During translation initiation, the small ribosomal subunit locates a specific segment in mRNA known as the translation-initiation region (Fig. 1a)1. 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. AminoacyltRNA is then delivered to the A site by the EF-Tu–GTP ternary complex. Formation of the first peptide bond and translocation represents the transition from the initiation step to the Alexander Mankin is at the Center for Pharmaceutical Biotechnology, The University of Illinois, 900 S. Ashland Ave., Rm. 3056, Chicago, Illinois 60607, USA. e-mail: [email protected] 858 a SD E P A mRNA SD E P A SD E P A Small subunit fMet-tRNA Subsequent steps of translation Large subunit Kasugamycin b E PA SD E P A SD x Initiation is blocked c E P A E P A Subsequent steps of translation d E P A E P A Translation can proceed Figure 1 Inhibition of translation initiation in bacteria by kasugamycin. (a,b) Kasugamycin inhibits translation initiation on mRNA with a 5ʹ leader sequence. a depicts bacterial translation initiation on 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 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 preventing efficient binding of initiator tRNA. (c,d) Translation initiation on leaderless mRNA may start with binding of the complete ribosome to the mRNA 5ʹ end (c) and is less kasugamycin sensitive. As kasugamycin overlaps with only one mRNA nucleotide and initiator tRNA binding is stabilized by its interaction with the 50S ribosomal subunit, initiation can proceed even in the presence of the drug (d). VOLUME 13 NUMBER 10 OCTOBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY © 2006 Nature Publishing Group http://www.nature.com/nsmb NEWS AND VIEWS elongation step of translation. The translationinitiation region in mRNA is composed of two elements: the initiator AUG codon and a ShineDalgarno region—a short nucleotide sequence complementary to the 3ʹ end of the small ribosomal subunit RNA2. These two elements are separated in mRNA by a short, 5- to 9-nucleotide spacer. In the context of the initiation complex, this mRNA spacer traverses the vacant exit (E) site, which in the elongating ribosome is temporarily occupied by deacylated tRNA. Kasugamycin has long been known to inhibit binding of initiator fMet-tRNA to the mRNAprogrammed small ribosomal subunit during translation initiation3. The conventional view of the mode of kasugamycin action came from genetic and biochemical studies. RNA footprinting experiments showed that kasugamycin protects two nucleotides in the rRNA of the Escherichia coli small ribosomal subunit, A794 and G926. Mutations at these positions confer resistance to the drug. In the classic footprinting experiments of Moazed and Noller4, positions A794 and G926 were also found to be protected by tRNA bound in the ribosomal P site. Therefore, it was perfectly reasonable to assume that kasugamycin interferes with initiation of translation by directly hindering binding of peptidyl-tRNA. However, two papers that appear on pages 871 and 879 of this issue show that this view requires substantial revision. Two independent teams used crystallography to understand where kasugamycin binds the ribosome and how it inhibits protein synthesis5,6. Whereas Schluenzen et al.5 soaked the drug into crystals of the Thermus thermophilus small ribosomal subunit, Schuwirth et al.6 made use of the complete E. coli ribosome, whose structure had recently been reported7. In spite of the differences in the source and composition of the ribosomes as well as the crystallization conditions, the structures (solved at 3.35 Å and 3.5 Å in refs. 5 and 6, respectively) yield similar conclusions about the position of kasugamycin in the bacterial ribosome. In its main site, the drug is bound in the cleft between the head and the platform of the small ribosomal subunit. This placement of kasugamycin fits well with the genetic and biochemical evidence, as the drug makes direct contact with A794 and G926. Furthermore, it is now clear that mutation of either of these two nucleotides directly affects the drug’s binding. The biggest surprise is that kasugamycin does not clash with the P-site tRNA. The minimal distance between the drug and the nearest position of the tRNA in the ribosomal P site is 6–7 Å, much too far for direct interference. Instead, kasugamycin is located in the path of mRNA in the ribosome. Comparison of the available crystallographic structures shows that kasugamycin overlaps with the position of three nucleotides of mRNA: the last two nucleotides of the E-site codon and the first nucleotide of the P-site codon (Figs. 1 and 2). Therefore, kasugamycin must inhibit binding of the fMet-tRNA to the 30S initiation complex only indirectly. This distinguishes kasugamycin from edeine, a chemically different inhibitor of translation initiation, which interacts with the same ribosomal site (Fig. 2)8. Edeine directly clashes with fMet-tRNA in the ribosomal P site8,9. Thus, two drugs interacting with almost the same site in the ribosome have markedly different mechanisms of action. In principle, kasugamycin might either prevent mRNA binding to the small ribosomal subunit or, alternatively, displace mRNA from its normal path. Though some weak competition between kasugamycin and mRNA was observed5, the most probable scenario is that the drug repositions mRNA in the initiation complex, leading eventually to a less efficient interaction of the initiator tRNA with the AUG codon. The action of kasugamycin clearly depends not only on its clash with the first nucleotide of the mRNA initiator AUG codon in the P site but also on displacement of the mRNA from the E site. Indeed, Schuwirth et al.6 show that inhibition of protein synthesis by kasugamycin depends on the identities of the two mRNA nucleotides that immediately precede the AUG codon and that therefore occupy the E site during translation initiation. This result corresponds well to reports that synthesis of different proteins in the cell is inhibited to different extents by kasugamycin10,11. Thus, the ribosomal E site and its possible interaction with the mRNA spacer connecting the Shine-Dalgarno sequence and the AUG codon may be important not only in elongation but also in initiation of translation. The overlap of the kasugamycin-binding site with the mRNA segment upstream of the AUG codon sheds light on another peculiarity of the drug’s action. It has long been known that kasugamycin inhibits translation of mRNAs equipped with a Shine-Dalgarno sequence but is markedly less efficient in inhibiting translation of leaderless mRNAs, which begin with the initiator AUG codon at their extreme 5ʹ end. Initiation of translation of such mRNAs may proceed through direct binding of mRNA and initiator fMet-tRNA to the 70S ribosome12,13. A less severe clash of the drug with the leaderless mRNA is probably one reason why kasugamycin more weakly inhibits translation of leaderless mRNAs. In addition, fMet-tRNA binding is enhanced in the 70S ribosome compared to the 30S initiation complex, owing to interaction of the tRNA with the large ribosomal subunit, which may help over- NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 10 OCTOBER 2006 tRNA anticodon Pct Edn Ksg2 Ksg1 E site P site Figure 2 The binding sites of two kasugamycin molecules (Ksg1 and Ksg2) on the T. thermophilus small ribosomal subunit overlap with the sites of action of edeine (Edn) and pactamycin (Pct). Green, mRNA; orange, anticodon stem-loop of P-site tRNA. Figure courtesy of Daniel Wilson. come its partial destabilization resulting from the displacement of mRNA by kasugamycin. Though the two new structures of kasugamycin–ribosome complexes are rather similar, there is one important difference. Whereas only one kasugamycin molecule is seen bound to the E. coli 70S ribosomes, two molecules of kasugamycin are clearly bound to the T. thermophilus small ribosomal subunit. In the T. thermophilus structure, the second molecule of kasugamycin binds right next to the first one—also on the path of mRNA through the E site (Fig. 2). The finding of the second kasugamycin binding site is puzzling. Studies of resistance mutations clearly show that the first site is the main site of kasugamycin action: all the known mutations are expected to affect binding of a kasugamycin molecule to that site. Nevertheless, the location of the second site is provocative, as it almost precisely coincides with the site of action of another antibiotic, pactamycin14 (Fig. 2). Pactamycin has long been considered an inhibitor of translation initiation, though more recent studies have shown that it interferes with translocation9. Interestingly, like kasugamycin, pactamycin may have specific effects on different mRNA sequences in the 30S subunit E site9. It is difficult to believe that kasugamycin could have evolved to bind two structurally different ribosomal sites. However, it is possible that, like the peptidyl-transferase center of the ribosome which can accommodate a variety of chemically diverse drugs, the second kasugamycin-binding site is generally favorable for binding of small molecules. Finding other compounds that may act upon this site could be an exciting challenge. 1. Gualerzi, C.O. & Pon, C.L. Biochemistry 29, 5881– 5889 (1990). 2. Shine, J. & Dalgarno, L. Nature 254, 34–38 (1975). 3. Poldermans, B., Goosen, N. & Van Knippenberg, P.H. 859 NEWS AND VIEWS J. Biol. Chem. 254, 9085–9089 (1979). 4. Moazed, D. & Noller, H.F. J. Mol. Biol. 211, 135–145 (1990). 5. Schluenzen, F. et al. Nat. Struct. Mol. Biol. 13, 871– 878 (2006). 6. Schuwirth, B.S. et al. Nat. Struct. Mol. Biol. 13, 879– 886 (2006). 7. Schuwirth, B.S. et al. Science 310, 827–834 (2005). 8. Pioletti, M. et al. EMBO J. 20, 1829–1839 (2001). 9. Dinos, G. et al. Mol. Cell 13, 113–124 (2004). 10. Okuyama, A. & Tanaka, N. Biochem. Biophys. Res. Commun. 49, 951–957 (1972). 11. Kozak, M. & Nathans, D. J. Mol. Biol. 70, 41–55 (1972). 12. Balakin, A.G., Skripkin, E.A., Shatsky, I.N. & Bogdanov, A.A. Nucleic Acids Res. 20, 563–571 (1992). 13. Moll, I., Hirokawa, G., Kiel, M.C., Kaji, A. & Blasi, U. Nucleic Acids Res. 32, 3354–3363 (2004). 14. Brodersen, D.E. et al. Cell 103, 1143–1154 (2000). Søren Lykke-Andersen & Torben Heick Jensen Eukaryotic transcriptomes are considerably larger than estimated from simple gene counts. However, much of this ‘excess’ RNA is immediately cleared from cells. Two recent studies reveal that so-called cryptic unstable transcripts constitutively transcribed from the yeast genome are rapidly eliminated in a process that couples transcription termination to RNA degradation. Cells harbor numerous surveillance pathways working to ensure genome, transcriptome and proteome integrity. The need for transcriptome quality control has recently been underscored by the realization that RNA polymerase II (RNAPII) transcription activity is extremely widespread and can be measured from areas of eukaryotic genomes whose expression has not been predicted by any judicious gene-annotation algorithm1. Some of the resulting products are important functional non–protein-coding RNAs, whereas others are presumably the outcome of anarchistic RNAPII behavior creating transcriptional noise, which has to be identified and eradicated by transcriptome surveillance. An illustrative example comes from the yeast Saccharomyces cerevisiae, where studies published last year identified a set of hitherto undiscovered RNAPII-dependent transcripts2. These RNAs, found to be widespread in the yeast genome, were dubbed cryptic unstable transcripts (CUTs), as their abundance is generally below the detection limit in wild-type cells. Indeed, CUTs were only convincingly revealed after their stabilization in strains deficient for RNA-surveillance factors, such as the nuclear exosome component Rrp6p or the poly(A) polymerase Trf4p from the exosomeactivating TRAMP complex2,3. In two new studies, the Corden and Libri laboratories further dissect the pathway of CUT decay and intriguingly show the involvement of factors normally engaged in transcription termination and 3ʹ-end formation of a separate class of RNAPII transcripts, the small nuclear and Søren Lykke-Andersen and Torben Heick Jensen are at the Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology, University of Aarhus, Aarhus, Denmark. e-mail: [email protected] 860 a RNAPII b mRNA c snoRNA CTD CUT C/D or H/ACA box 5′ RNA Nrd1–Nab3 binding site 5′ 5′ Nrd1–Nab3 Pap AAA AAA Exosome Exosome– TRAMP Figure 1 Processing and degradation of 3ʹ ends of three different RNAPII transcripts. (a) Transcription termination of mRNA-encoding genes is closely linked to the cleavage and polyadenylation reaction (mediated by Pap, light green) and requires the CTD of RNAPII. The mature poly(A) tail (AAA) is immediately covered by poly(A)-binding proteins (yellow oval), which protect the mRNA from decay by the nuclear exosome. (b) The snoRNA genes that constitute independent transcription units are terminated via the Nrd1p–Nab3p complex, which recognizes specific binding sites in the emerging transcript. The complex also interacts with the RNAPII CTD and, by a yet-undiscovered mechanism, facilitates RNAPII transcription termination. In a subsequent reaction, the nuclear exosome trims the snoRNA 3ʹ end down to one of two types of RNA structures (the C/D or H/ACA box) bound by protective proteins (blue). (c) CUTs are also terminated via the Nrd1p–Nab3p pathway. However, in this case, the nuclear exosome, in conjunction with its activator, TRAMP, degrades the transcripts completely. small nucleolar RNAs (snRNAs and snoRNAs; Fig. 1)4,5. In addition to changing our conventional way of categorizing RNAPII products, these observations also suggest a way in which novel RNP-encoding units might evolve. Quality control of RNPs and their constituent RNAs takes place in both the cell nucleus and cytoplasm, and, most often, error detection is followed by rapid disposal and subsequent recycling of reusable parts. While known cytoplasmic RNA quality-control pathways are linked to translation, the monitoring of transcript quality in the nucleus is coupled to the multiple maturation processes that all classes of RNAs undergo in this compartment6,7. The nuclear exosome is an integral component of nuclear RNA maturation and surveillance pathways. It has dual roles, as it not only assists in the controlled maturation of certain RNAs by trimming their 3ʹ ends but also can execute the complete 3ʹ→5ʹ degradation of aberrant RNAs (Fig. 1). TRAMP, a recently discovered nuclear exosome–associated protein complex, facilitates the function of the nuclear exosome, presumably by appending oligo(A) tails onto destruction-tagged RNA substrates on which 3ʹ→5ʹ exonucleolytic activity is otherwise blocked2,8,9. Having previously shown that CUT decay is exosome–TRAMP dependent, Thiebaut and colleagues set out to examine details of the surveillance of these transcripts by studying the model substrate NEL025c. This RNA is readily detectable when the nuclear exosome VOLUME 13 NUMBER 10 OCTOBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY Ebbe Sloth Andersen © 2006 Nature Publishing Group http://www.nature.com/nsmb CUT it out: silencing of noise in the transcriptome