tural Animal Solutio “There is a lot of truth in the naturopathic belief that all diseases start in the gut. Homoeostasis depends on the dynamic balance between gut microfl ora, the mucosal barrier and the immune system” Herbs and Nutrients for fermentation of non-digestible dietary a Healthy Gut Fermentation of carbohydrates is a major source
Gkh583 2594.2597Published online May 11, 2004
2594±2597 Nucleic Acids Research, 2004, Vol. 32, No. 8 Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coliMaria M. Anokhina1, Andrea Barta2, Knud H. Nierhaus3, Vera A. Spiridonova4 andAlexei M. Kopylov1,4,* 1Department of Chemistry, Moscow State University, 119992 Moscow, Russian Federation, 2Institute of Biochemistry, University of Vienna, Vienna Biocenter, A1030 Vienna, Austria, 3Max-Planck Institute for Molecular Genetics, D-14195 Berlin-Dahlem, Germany and 4A.N. Belozersky Institute of Physical Chemical Biology, Moscow State University, 119992 Moscow, Russian Federation Received February 5, 2004; Revised March 22, 2004; Accepted April 14, 2004 At present, three sets of data on four different types of Tetracycline blocks stable binding of aminoacyl- bacteria are available: solution studies (E.coli), X-ray analysis (T.thermophilus) and genetic data (Propionibacterium acnes, tRNA to the bacterial ribosomal A-site. Various tetra- cycline binding sites have been identi®ed in crystals Recently, X-ray analyses by two groups (9,10) have of the 30S ribosomal small subunit of Thermus resolved the structure of Tc in complexes with crystals of thermophilus. Here we describe a direct photo- the 30S ribosomal subunit of T.thermophilus. One group (10) af®nity modi®cation of the ribosomal small subunits found six Tc binding sites named Tet-1 to Tet-6 (Fig. 3). The of Escherichia coli with 7-[3H]-tetracycline. To select other group (9) identi®ed two sites: one is almost identical to for speci®c interactions, an excess of the 30S sub- Tet-1, and the other one is close to Tet-5. This ®nding units over tetracycline has been used. Primer exten- indicates seven different putative binding sites for Tc inter- sion analysis of the 16S rRNA revealed two sites of acting with the crystals of ribosomal small subunit of the modi®cations: C936 and C948. Considering T.thermophilus. It should be mentioned that the complexes available data on tetracycline interactions with the of Tc were formed by soaking with the ribosomal crystals of prokaryotic 30S subunits, including the presented T.thermophilus, although nothing is known about interactions data (E.coli), X-ray data (T.thermophilus) and In contrast to many translational inhibitors where resistance genetic data (Helicobacter pylori, E.coli), a second markers on the ribosomes have been known for decades, high af®nity tetracycline binding site is proposed genetic data on ribosomal mutations conferring resistance within the 3¢-major domain of the 16S rRNA, in against Tc have been reported only recently. Ross et al. (11) addition to the A-site related tetracycline binding and Trieber and Taylor (12) have mapped mutations in the 16S rRNA from Tc-resistant natural bacterial isolates. In one case, the location of mutated nucleotide G1058C of the helix H34 of the 16S rRNA is close to the Tet-1 site (9,10), which is in agreement with the above-mentioned view that this site is Tetracycline (Tc) is a major member of a group of antibiotics responsible for the inhibition of the A-site stable occupation.
with a broad spectrum of activity, which is widely used in Recently Trieber et al. (12) have published a new set of data medicine and veterinary science to treat bacterial infections, as on mapping of Tc-resistant mutants in 16S rRNA which could well as for food production (1). After penetration into bacterial be attributed (by us) to the Tet-4 site (10).
cells, Tc interacts with ribosomes and inhibits protein We set out to collect data concerning the binding sites on biosynthesis. The drug blocks stable binding of aminoacyl- the ribosomal 30S subunit of E.coli in solution. We have tRNA to the A-site of ribosomes (2±5). But despite intensive applied one of the widely used methods, photo-af®nity studies for over more than 50 years, the exact molecular modi®cation, to map Tc-binding site(s) on the ribosomes.
mechanism of Tc interactions with bacterial ribosomes is still The Tc molecule has two uncoupled conjugated bond systems: ring A and rings B-C-D (Fig. 1A). The two ring X-ray studies of functional ribosome complexes of Thermus systems are the reason for two peaks in the absorption thermophilus have led to a quantum leap in our understanding spectrum of Tc (Fig. 1B). Irradiation of the Tc±ribosome of mechanisms of protein synthesis. Three tRNA binding sites complex with light of 365 nm excites the Tc molecule (13) and have been mapped, the A, P and E sites (6±8). A localization yields a covalent bond with reactive groups of the ribosome in of Tc binding site(s) on Escherichia coli ribosomes was therefore an essential step towards an understanding of the Goldman and colleagues (15,16) were the ®rst to use direct photo-af®nity Tc-modi®cation of the 30S ribosomal subunit of *To whom correspondence should be addressed. Tel: +7 095 939 3143; Fax: +7 095 939 3181; Email: firstname.lastname@example.org Nucleic Acids Research, Vol. 32 No. 8 ã Oxford University Press 2004; all rights reserved Nucleic Acids Research, 2004, Vol. 32, No. 8 2595 Figure 2. Primer extension analysis of the 16S RNA using the primer CGACAGCCATGCAGCACC complementary to G1047±G1064 of the 16S rRNA. Separation on an 8% polyacrylamide-urea gel demonstrates reverse transcriptase primer extension stops at positions A937 and A949, caused by modi®cation of the 16S rRNA with Tc. The fragment of the 16S rRNA sequence A918±U957 is shown. Line 1, the 16S rRNA isolated from the irradiated Tc-30S subunit complex; line 2, the 16S rRNA isolated from irradiated 30S subunits (no Tc); line 3, the 16S rRNA isolated from 30S 3 mM MgAc2, 150 mM NH4Cl, 4 mM mercaptoethanol, 0.05 mM spermin, 2 mM spermidin, which has been optimized for functional assays (19±21). The mixture of 1 mM of 7-[3H]- Tc and 2 mM of 30S subunits was incubated in 1 ml of the binding buffer for an additional 15 min at 37°C.
The extent of complex formation was measured by the ®lter-binding assay as described (14): an aliquot was ®ltered through nitrocellulose membrane (0.45 mm, Sartorius Figure 1. (A) Structure of Tc complex with Mg2+ (25). (B) Absorption 113-06-N, Germany). After drying, the amount of bound Tc was counted in 5 ml of toluene scintillation ¯uid (GS-106, Russia), using a Tracor Analytic scintillation counter (France).
For the modi®cation, a 250 W high-power Hg arc lamp E.coli. In addition to some nonspeci®cally modi®ed proteins: (DRSh-250, PhysPribor, Russia) has been used with the main S18, S4, S14 and S13 (15), the protein S7 turned out to be the emission maximum near 365 nm. Samples were irradiated for major target (16). Using a more advanced approach, Oehler 2.5 min at 0°C, in a 313 nm cut-off plastic cuvette with 10 mm et al. (17) also found a modi®cation of S7, as well as the 16S optical path (Sarstedt, Germany), which was positioned 25 cm Because both above mentioned groups of researchers had used a large molar excess of Tc over the ribosome, which Primer extension analysis of the 16S rRNA modi®cations could promote additional nonspeci®c binding [as was shown The 16S rRNA was isolated from the irradiated Tc±30S earlier for Tc binding to transcriptional repressor protein ribosome complex by standard phenol extraction, and was TetR(D) (14,18)], we paid particular attention to the Tc/ used for reverse transcriptase primer extension analysis as ribosome ratio during complex formation. Under selected conditions, the photolysis of the complex of Tc with 30S subunit yields about equal modi®cations of both proteins and the 16S rRNA. Here we report the analysis of the 16S rRNA The key points of this study are that: (i) the binding of Tc was performed with very active E.coli ribosomes (19), (ii) the buffer used is optimal for the analysis of ribosomal functions (19±21), and (iii) an excess of the 30S subunits over Tc has 30S ribosomal subunits of E.coli were isolated as described Our preliminary data on Tc interactions with E.coli (19). 7-[3H]-Tc with a speci®c activity of 37 GBq/mmol was ribosomes, using nitrocellulose-binding assay, have revealed that the extent of Tc binding to either 70S ribosomes or 30S subunits is about the same. In addition, it turned out that for a high yield of complex it is not obligatory to use a large excess For the complex formation, 30S subunits were pre-incubated of Tc over the ribosome, but just proper concentrations of for 10 min at 37°C in the buffer: 20 mM HEPES±KOH pH 7.6; the components ([Tc] = 1 mM, [30S] = 2 mM), close to the 2596 Nucleic Acids Research, 2004, Vol. 32, No. 8 nucleotide was taken as the following nucleotide in the 16S Primer extension analysis of one region of the 16S rRNA, where Tc modi®ed nucleotides were found, is shown in Figure 2 (line 1); the sequence interval was U920-A1046. Two modi®ed nucleotides, C936 and C948, have been clearly and reproducibly detected. Only two stops have been selected as they are the only ones which do not have any detectable counterparts in the control lines 2 and 3 (Fig. 2). The differences in the modi®cation pattern from the previous results (17) are probably due to the fact that here much lower (sub-stoichiometric) amounts of Tc were used.
DISCUSSIONTwo groups (9,10) have identi®ed two and six Tc binding sites, respectively, for crystals of 30S subunits from T.thermophilus. This therefore presents a problem in assigning one of the crystallographically determined sites for one type of the ribosomes (T.thermophilus) to the biologically relevant inhibitory site(s) for the other type of ribosomes (E.coli).
In a simple way, it could be expected that a single inhibitory functional site is close to the ribosomal A-site. The location of the Tet-1 site (9,10) is in good agreement with a conventional view that this site is responsible for drug interference with the aminoacyl-tRNA accommodation within the A-site (5). In addition, in vivo genetic studies of different natural bacterial Figure 3. Putative sites of Tc interactions with the 16S rRNA within isolates (11,12), as well as some indirect data (5), also indicate crystals of the 30S ribosomal subunit of T.thermophilus according to PDB 1I97 (10). PDB data were analyzed with Swiss PDB Viewer 3.6b3 (http:// that there could be at least one more binding site for Tc, cn.expasy.org/spdbv). The 16S rRNA sequence numbering is according to though its location is not yet clear. The solution data, E.coli. G942 corresponds to in vivo genetic data (12), C936 and C948 data published earlier for E.coli ribosomes, as discussed in the from this publication. The ribosomal small subunit interface with six Introduction, could not resolve this ambiguity, probably due to putative Tc binding sites is on the left. (A) The extracted structure of the the use of a large excess of Tc over ribosomes in the main sub-domain of the 3¢-end major domain of the 16S rRNA (26±28) showing RNA in the dark gray ribbons, and S7 protein in light gray cylinders. Tet-4, Tet-6 and G942, C936, C948 are shown. Orientation of the In this study, we have revealed a second high af®nity Tc sub-domain is the same as for the subunit. (B) Space-®lled Tet-4 and Tet-6 binding site within the 3¢-major domain of the 16S rRNA of (black), and the 16S rRNA nucleotides (gray) are depicted with the same the 30S subunit of E.coli ribosome, close to the Tet-4 site, in orientation as in (A). (C) The image is depicted at an orientation, different from that in (B), to show the distances (AÊ) in more detail.
addition to the A-site related Tc binding site Tet-1.
C936 belongs to a single-stranded region of the 16S rRNA connecting helices H28 and H29, and C948 belongs to the helix H30 of the 16S rRNA. These positions are located close corresponding value of the binding constant (2 Q 106 M±1) to the Tc binding sites Tet-4 and Tet-6 (10). Our computer annotation of available X-ray data [PDB 1I97 (10)] has For photo-af®nity modi®cation, the Tc/30S subunit ratio revealed the following picture (Fig. 3). Tc could modify C936 was 1:2; 45% of the input Tc was bound to 30S subunits under from either/both Tet-4 and Tet-6 binding sites, which are at an this condition. The photo-af®nity reaction for the [3H]-Tc-30S equal distance of about 10 AÊ from C936. On the other hand, subunit complex was triggered by irradiation at a wavelength both C936 and C948 could be modi®ed simultaneously, if of 365 nm, for 2.5 min at 0°C, which represents a short Tet-4 was occupied as the only site. In this case, the distances irradiation time compared with earlier studies (16). In from Tet-4 to C936 and C948 are 9.8 and 14.2 AÊ, respectively.
addition, the buffer used contained mercaptoethanol to avoid The exact mechanism of Tc photolysis is not known in light-independent incorporation of Tc photo-products (13,16).
detail (13). Therefore, the probe±target distance for modi®- It turned out that the covalently linked [3H]-Tc-label was cation with excited Tc molecules is not known either. The equally distributed between the 16S rRNA and the ribosomal af®nity modi®cation event does not necessarily mean that proteins, as has been previously described (17). The 16S reactive residues are in direct contact. For example, Lancaster rRNA was isolated and analyzed by primer extension (17,22).
et al. (24) have revealed that the distribution of probe±target The chosen set of primers allows scanning of the entire 16S distances for directed hydroxyl radical cleavages measured rRNA sequence, except the very 3¢-end region. The 16S from the S8-16S rRNA models might be within the range of rRNAs both from 30S subunits irradiated without Tc and from 20 AÊ, and even more. If one takes into account the size of the non-irradiated 30S subunits were used as controls for identi- Tc molecule of about 6 Q 12 AÊ, the distances determined from ®cation of random stops on the RNA template (Fig. 2, lines 2 the established Tc binding sites seem to be reasonable. In and 3, respectively). When a stop was observed, the modi®ed addition, Tc binding in solution with 30S subunits might Nucleic Acids Research, 2004, Vol. 32, No. 8 2597 induce subtle changes in this binding region, which could not 9. Brodersen,D.E., Clemons,W.M.,Jr, Carter,A.P., Morgan-Warren,R.J., be observed by binding to the rigid crystals. This would bring Wimberly,B.T. and Ramakrishnan,V. (2000) The structural basis for the Tc even closer to the modi®ed nucleotides.
action of the antibiotics tetracycline, pactamycin and hygromycin B on the 30S ribosomal subunit. Cell, 103, 1143±1154.
In our previous publication (17), it was shown that a large 10. Pioletti,M., Schlunzen,F., Harms,J., Zarivach,R., Gluhmann,M., excess of Tc could modify a different set of 16S rRNA Avila,H., Bashan,A., Bartels,H., Auerbach,T., Jacobi,C. et al. (2001) nucleotides: G693, G1300 and G1338. There is no direct Crystal structures of complexes of the small ribosomal subunit with correlation between binding to the particular site and possible tetracycline, edeine and IF3. EMBO J., 20, 1829±1839.
yield of cross-linking within the site. Therefore, if the excess 11. Ross,J.I., Eady,E.A., Cove,J.H. and Cunliffe,W.J. (1998) 16S rRNA mutation associated with tetracycline resistance in a Gram-positive of Tc modi®es more reactive nucleotides in some other sites, bacterium. Antimicrob. Agents Chemother., 42, 1702±1705.
then the modi®cations described might have been masked.
12. Trieber,C.A. and Taylor,D.E. (2002) Mutations in the 16S rRNA genes Our suggestion that the modi®cation could occur from the of Helicobacter pylori mediate resistance to tetracycline. J. Bacteriol., Tet-4 binding site perfectly correlates with recent in vivo 13. Beliakova,M.M., Bessonov,S.I., Sergeyev,B.M., Smirnova,I.G., genetic data for natural isolates of Tc-resistant strains of Dobrov,E.N. and Kopylov,A.M. (2003) Rate of tetracycline photolysis H.pylori or for arti®cially created strains, revealing that a during irradiation at 365 nm. Biochemistry (Mosc.), 68, 182±187.
deletion of G942 (helix H29) of the 16S rRNA confers 14. Beliakova,M.M., Anokhina,M.M., Spiridonova,V.A., Dobrov,E.N., moderate Tc resistance up to 8-fold (12). Figure 3 shows that Egorov,T.A., Wittmann-Liebold,B., Orth,P., Saenger,W. and G942 is in very close proximity to Tet-4 (2.7 AÊ).
Kopylov,A.M. (2000) A direct photo-activated af®nity modi®cation of tetracycline transcription repressor protein TetR(D) with tetracycline.
We can reconcile our observations in the following way.
The ®rst binding site can be ascribed to the well accepted A- 15. Goldman,R.A., Cooperman,B.S., Strycharz,W.A., Williams,B.A. and site related Tc binding site, Tet-1. And in a separate set of Tritton,T.R. (1980) Photoincorporation of tetracycline into Escherichia experiments on photo-af®nity modi®cation of the 30S subunit, coli ribosomes. FEBS Lett., 118, 113±118.
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