Structural information on the binding of antibitiocs to the 30S subunit has been obtained by two groups so far. Most of the data have been published by V. Ramakrishnan's group at MRC, who solved the crystal structure of the complex with Spectinomycin, Streptomycin and Paromomycin (all three bound simultaneously). Shortly afterwards (Dec. '00) the structures of complexes with Tetracycline, Pactamycin, and Hygromycin B were released. In April 2001 we (MPG) finally managed to publish structures of complexes with Edeine and Tetracycline (and the C-terminal domain of IF3). Edeine turned out to be rather similar to pactamycin, with some crucial differences.
An overview about all the binding sites is shown below, and a little more detail (at least for edeine and tetracycline) further down ...
The binding site of edeine is close to the 3' end of 16S rRNA, where the shine-dalgarno part of the mRNA is supposed to bind. The beta-tyrosine moiety however points towards the P(eptidyl)-site of the 30S subunit. As a consequence, mRNA binding is hindered and the P-site tRNA can not bind in a productive way. Presumably, edeine would also block the progression of mRNA around the neck of the 30S subunit. The c-terminal domain of IF3-C, is also involved in the recognition of the shine-dalgarno sequence and in processing of leaderless mRNA: As we could show (though at low resolution), IF3-C or more accurare the so called linker, which connects the N-terminal and the C-terminal domains, occupies the same region in the 30S subunit as edeine, so that the antibiotic might also interfere with proper IF3 binding.
Position of edeine in the 30S
Remarkable is a conformational change in 16S rRNA induced by edeine, which results in the formation of a new inter-helix base pair beween helices 23 and 24. This new base pair creates a strong brodge between two sides of the platform, which suppresses independent movements. Since biochemical data and images obtained by electron microscopy imply that large conformational changes occur and are required for the formation of the initiation complex, its plausible that the induction of the base pairing is actually the most important part of the antimicrobial activity of edeine.
A closer view of the image above: The electron density of the newly created base pair between helices 23 and 24.
A tiny animation showing the conformations before and after edeine binding.
were among the very first braod spectrum antibiotics, acting bacteriostatic against a wide range of different bacteria.
Tetracyclines tend to accumulate in bones and teeths.
The strong fluorescence of some tetracyclines invoked some amazing stories:
One of my favorite ones is this one: a clinical doctor comning home late from work noticed a rather bright light coming from the bin. Aliens ? Atomic waste ? Dinner !
The fluorescence is sometimes also utilized (or misused ?) for incredible application, which certainly contribute to the high level resistances against tetracyclines -->see Resistence.
So how does tetracycline act on the ribosome?? Early biochemical studies were rather contradictory, suggesting more than just a single binding site. But the observation that mutations of a single 16S rRNA base leads to resistance against tetracyclines indicated, that probably only one of those sites is responsible for the antimicrobial activity. And indeeed, we found 6 binding sites in the structure of the 30S-tetracycline complex, but only one molecule which is obviously inhibiting protein synthesis.
This molecule binds - with the help of a magnesium ion - to the sugar-phosphate backone (light blue) of helix 34, just in the vicinity of the A-site tRNA docking site.
Biochemical data indicated, that binding of aa(aminoacytylated)-tRNA is not inhibited but the movement of the tRNA into its proper site (with respect to the 50S subunit) is suppressed, such that peptide bond formation becomes impossible for the targeted ribosome.
As mentioned before, we located 5 other molecules per 30S subunit. One tetracycline molecule, which was also detected by the group in Cambridge, binds in a tight pocket between protein S17 and helices 11 and 27. The influence of S17 is probably not crucial, since this part is specific to termophilic bacteria. However, helix 27, which is sometimes called the switch helix, is a hot spot of the decoding mechanism. H27 is supposed to acquire different base pairings depending on the stage of decoding (or translocation of tRNA). Tetracycline rigidifies this region by creating a link between H11 and H27. The binding of tetracycline to this site might therefore - as a side effect - prevent the switch of the base pairing scheme. It apparently doesn't stop the ribosome from producing proteins, but it might well render the process rather inefficient.
The other four sites do not appear to have an obvious effect on the function of the ribosome, and are therefore only briefly described:
345,6
Position 3 binds to protein S4, one of the early assembly proteins. It might have an effect on the assembly of the 30S subunit, but thats just an idea.
Molecule number 4 just percolates into the groove of helix 40, without any obvious effect. Molecules 5 and 6 finally bind in the upper part of the 30S subunit, the neck resp. the head. No. 5 contacts helix 28 and protein S7, no. 6 mainly to the long extension of protein S9. Their contribution to any inhibitory action is more than questionable ... however, the 6 binding sites explain well the various biochemical data, which implicated rather different regions of the 30S subunit to be involved in tetracycline binding, which all turned out to be correct, though not exactly the way expected ....
Paromomycin, Streptomycin, Spectinomycin, Pactamycin, Hygromycin B
Paromomycin (1FJG), is an aminoglycoside, which increases the error rate of the ribosome. It binds in the major groove of helix 44 of 16S rRNA just 'below' the decoding site. The major action of paromomycin derives from a conformational change in helix 44. In the presence of this compounds, two rRNA bases of 16S rRNA appear to flip out pointing directly into the A-site. It was proposed, that the bases will acquire this orientation only in the presence of a cognate A-site tRNA molecule, but not when a non-cognate tRNA is bound to the A-site. Paromomycin would hence increase the affinity of non-cognate tRNA's for the A-site, and this way render the ribosome error prone.
Streptomycin (1FJG)
was shown to make the ribosome error prone by affecting the proof reading step. It binds to four nucleotides of 16S rRNA and a single amino acid of protein S12. Streptomycin probably locks the ribosomes in the so called 'ram-state' (Ribosome Ambiguity Mutation), which can compensate for hyper accuracy induced by certain mutations of S12, presumably by strongly increasing the affinity for non-cognate tRNA molecules in the A-site.
Spectinomycin (1FJG)
binds in tje minor groove at one end of helix 34, in the vicinity of helix 28 and protein S5. Though the mechanism of this antibiotic remained a bit unclear, Spectinomycin probably hinders the rotation of the head, either directly or by fixing helix 34 in a specific conformation. As a consequence, translocation of peptidyl-tRNA from the A-site to the P-site is inhibited, as proposed by biochemical data....
Pactamycin (1HNX)
is like Edeine a so called universal antibiotic, which inhibits ribosomal protein synthesis in all organisms. Pactamycin binds in a single site on the 30S in the upper part of the platform, very close to the cleft in the subunit that is responsible for binding of the three tRNA molecules. Though it was originally assumed that pactamycin binds primarily to the P-site, it turns out that it's located in the E-site of the 30S subunit. The mRNA is heavily displaced by pactamycin, by roughly 12Å for the last base of the E-site codon. Pactamycin therefore hinders productive binding of the initiator tRNA and passage of the mRNA through the ribosome, which consequently inhibits formation of the initiation complex. Of course, pactamycin would also precluce interactions of the mRNA with the E-site bound tRNA, but it appears unlikely that this has a major contribution to the antibiotic activity. Simply because neither pactamycin nor edeine bind to elongating ribosomes, but act only at the initiation stage, so that the E-site will never be occupied in the presence of these compounds.
has like all the other aminoglycosides a single site. It binds in the major groove at the very top of helix 44, in a highly sequence-specific manner. Though the mechanism of its activity is not obvious, it seem rather likely that hygromycin B inhibits a conformational transition required for translocation. This in turn would prevent movement of the A-site tRNA in the P-site, which agrees well with early biochemical data.
The structures of these five compounds, have been solved by V. Ramakrishnan and Co-workers, so we didn't want to go too much into details about work done by someone else. But more information about these antibiotics as well as a more detailed view on different comformational stages of the 30S subunit can be found on the Homepage of the Cambridge group.
Closely related to the activity of the antibiotics, or more precisely its lost activity, is the growing number of resistant pathogens. We collected some information about the (mis)use of antibiotics and resistances against them on a separate page.
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