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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