1 Max-Planck Arbeitsgruppe Ribosomenstruktur, Notkestr. 85, 22607 Hamburg,
Germany
2 Department of Structural Biology, Weizmann Institute, 76100 Rehovot,
Israel
* Author for correspondence (e-mail: harms{at}mpgars.desy.de)
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Introduction |
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Responsibility for the various steps of polypeptide synthesis is divided among the two ribosomal subunits (30S and 50S). The 30S subunit ensures fidelity of decoding by establishing accurate codon-anticodon interactions. The 50S subunit catalyses peptide bond formation and elongation of the nascent protein, further protecting the nascent chain by channelling it through the ribosomal exit tunnel, which links the peptidyl transferase centre to the bottom of the 50S subunit.
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Initiation |
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Edeine and pactamycin are universal antibiotics that affect protein
biosynthesis in all organisms. Both hamper the formation of the initiation
complex by displacing the mRNA (Brodersen
et al., 2000; Pioletti et al.,
2001
). Edeine also affects the positioning of peptidyl site
(P-site) tRNA and IF3 function. Evernimicin interacts with the tips of the 23S
rRNA helices 89 and 91, which bind IF2, and may prevent formation of the 70S
initiation complex (Belova et al.,
2001
).
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Decoding and A-site occupation |
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Many antibiotics target this step of the elongation cycle. Tetracycline,
the first broad-spectrum antibiotic discovered, binds to multiple sites in the
ribosome (Brodersen et al.,
2000; Pioletti et al.,
2001
). Binding to the A-site in the 30S subunit sterically hinders
the movement of the aa-tRNA so that it cannot simultaneously interact with the
decoding site in the 30S subunit and the peptidyl transferase centre in the
50S subunit. The remaining sites may affect the assembly of the ribosomal
particle but are probably not responsible for any inhibitory activity, since a
single mutation in the primary binding site confers tetracycline
resistance.
Aminoglycosides bind directly adjacent to the decoding site in the 30S
subunit, rendering translation highly inaccurate. Paromomycin, for example,
induces a conformational change that enhances the affinity of the A-site for
near-cognate tRNAs. Hygromycin B and neomycin act by freezing the tRNA in the
A-site (Brodersen et al.,
2000). Streptomycin stabilizes the so-called ram state of the 30S
subunit, in which its affinity for non-cognate tRNAs is increased
(Carter et al., 2000
).
Three antibiotics hamper positioning of the A-site tRNA by acting on EF-Tu:
pulvomycin seems to reduce the GTPase activity of EF-Tu and inhibits its
binding to tRNA; GE2270A blocks the tRNA-binding site on EF-Tu; and kirromycin
prevents the release of EF-Tu, thereby stalling the ribosome
(Hogg et al., 2002).
Several antimicrobial agents inhibit decoding or A-site occupation by
interacting with the 50S subunit. Sparsomycin, for example, binds in the core
of the peptidyl transferase centre (PTC) by stacking to the most flexible
nucleotide, which induces substantial conformational alterations
(Bashan et al., 2003) and
affects the correct positioning of both the A-site and P-site tRNAs
(Hansen et al., 2002
).
Linezolid, an oxazolidinone, increases frameshifting and stop codon
readthrough with nonsense suppression. It presumably binds in the vicinity of
the PTC at the interface between the ribosomal subunits, thereby affecting
initiation and elongation, but might also affect accuracy of decoding.
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Peptide bond formation |
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A variety of organisms produce antimicrobial compounds that target peptide bond formation. These are effective against many competing organisms because of the high phylogenetic conservation of the PTC; however, the originator is also attacked by its drug and must therefore develop resistance mechanisms, which are sometimes costly. Drugs inhibiting peptide bond formation act in three distinct ways: (1) by mimicking the substrate; (2) by blocking the path of the growing polypeptide chain; or (3) by alteration of tRNA positioning on the ribosome.
Puromycin is a universal antibiotic and a tool for investigating the
mechanism of peptide bond formation. Mimicking the aminoacylated end of the
aa-tRNA, it participates in peptide bond formation, but its non-hydrolysable
amide bond cannot be cleaved and peptidylpuromycin falls off the ribosome
(Nissen et al., 2000;
Bashan et al., 2003
).
Chloramphenicol, rarely used nowadays owing to its significant side effects,
acts at the same site in the PTC. It occupies the position of the amino acid
attached to the A-site tRNA, preventing peptide bond formation
(Schlünzen et al.,
2001
).
Lincosamides (clindamycin and lincomycin) interact with both the A-site and
P-site on the 50S subunit, hampering positioning of both tRNA molecules and
directly inhibiting peptide bond formation. Macrolides (e.g. erythromycin)
instead bind at the entrance of the ribosomal exit tunnel
(Schlünzen et al., 2001),
blocking the path of the nascent chain through the ribosome. This site is some
distance from the PTC; thus a polypeptide chain of 3-5 residues can be
produced before protein biosynthesis stalls. Semi-synthetic derivatives of
erythromycin such as carbomycin A or josamycin possess a long,
mycarose-isobutyrate extension of the desosamine sugar (the crucial functional
group of the macrolides) that protrudes into the PTC. These compounds thus
also interfere with the correct positioning of the P-site substrate and
directly affect peptide bond formation
(Hansen et al., 2002
).
Azithromycin, a rather new erythromycin derivative, binds to two sites in the
D. radiodurans 50S subunit
(Schlünzen et al., 2003
).
The two azithromycin molecules are in direct contact; so in this case a
cooperative effect might enhance its antimicrobial activity. Streptogramins
also have a dual inhibitory mechanism, but this derives from two unrelated
compounds: streptogramins A and streptogramins B. These enhance each other's
activity probably through a direct interaction. Streptogramins A destabilize
the binding of tRNAs to the A-site and P-site; streptogramins B presumably
occupy the tunnel for the nascent peptide chain similarly to the
macrolides. Macrolides, lincosamides and streptogramins B all fail to inhibit
MLSB-resistant strains possessing a single modification in the rRNA
of the PTC, which reflects the overlapping binding site of these
antibiotics.
The mechanism behind the antimicrobial activity of another group of compounds, the pleuromutilins (e.g. tiamulin or valnemulin), is unclear. Since they compete with carbomycin but not erythromycin, they might prevent correct positioning of the CCA ends of tRNAs for peptide transfer.
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Translocation and tRNA-release |
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Viomycin acts at a slightly earlier stage: it seems to hamper translocation
so that the A-site remains blocked. Viomycin has some additional effects, such
as induction of misreading, inhibition of the peptidyl transferase reaction
and inhibition of subunit dissociation during termination. Like many other
drugs, it appears to bind at the interface between the subunits, thus altering
the positioning of the A-site and P-site tRNAs, possibly affecting the
affinity of the 50S subunit for them. Spectinomycin, another aminoglycoside
antibiotic, is a rather rigid molecule. It binds to the head region of the 30S
subunit, which undergoes a conformational change during elongation.
Spectinomycin prevents this transition, inhibiting EF-G-catalysed
translocation of the peptidyl-tRNA from the A-site to the P-site
(Carter et al., 2000).
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Termination |
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References |
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