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Published online May 11, 2004
2594±2597 Nucleic Acids Research, 2004, Vol. 32, No. 8 DOI: 10.1093/nar/gkh583

Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coli
Maria M. Anokhina1, Andrea Barta2, Knud H. Nierhaus3, Vera A. Spiridonova4 and Alexei M. Kopylov1,4,*
Department 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
1

ABSTRACT Tetracycline blocks stable binding of aminoacyltRNA to the bacterial ribosomal A-site. Various tetracycline binding sites have been identi®ed in crystals of the 30S ribosomal small subunit of Thermus thermophilus. Here we describe a direct photoaf®nity modi®cation of the ribosomal small subunits of Escherichia coli with 7-[3H]-tetracycline. To select for speci®c interactions, an excess of the 30S subunits over tetracycline has been used. Primer extension analysis of the 16S rRNA revealed two sites of the modi®cations: C936 and C948. Considering available data on tetracycline interactions with the prokaryotic 30S subunits, including the presented data (E.coli), X-ray data (T.thermophilus) and genetic data (Helicobacter pylori, E.coli), a second high af®nity tetracycline binding site is proposed within the 3¢-major domain of the 16S rRNA, in addition to the A-site related tetracycline binding site. INTRODUCTION Tetracycline (Tc) is a major member of a group of antibiotics with a broad spectrum of activity, which is widely used in medicine and veterinary science to treat bacterial infections, as well as for food production (1). After penetration into bacterial cells, Tc interacts with ribosomes and inhibits protein biosynthesis. The drug blocks stable binding of aminoacyltRNA to the A-site of ribosomes (2±5). But despite intensive studies for over more than 50 years, the exact molecular mechanism of Tc interactions with bacterial ribosomes is still unknown in detail (5). X-ray studies of functional ribosome complexes of Thermus thermophilus have led to a quantum leap in our understanding of mechanisms of protein synthesis. Three tRNA binding sites have been mapped, the A, P and E sites (6±8). A localization of Tc binding site(s) on Escherichia coli ribosomes was therefore an essential step towards an understanding of the molecular mechanism of its action.

At present, three sets of data on four different types of bacteria are available: solution studies (E.coli), X-ray analysis (T.thermophilus) and genetic data (Propionibacterium acnes, Helicobacter pylori, E.coli). Recently, X-ray analyses by two groups (9,10) have resolved the structure of Tc in complexes with crystals of the 30S ribosomal subunit of T.thermophilus. One group (10) found six Tc binding sites named Tet-1 to Tet-6 (Fig. 3). The other group (9) identi®ed two sites: one is almost identical to Tet-1, and the other one is close to Tet-5. This ®nding indicates seven different putative binding sites for Tc interacting with the crystals of ribosomal small subunit of T.thermophilus. It should be mentioned that the complexes of Tc were formed by soaking with the ribosomal crystals of T.thermophilus, although nothing is known about interactions of Tc with thermophilic ribosomes. In contrast to many translational inhibitors where resistance markers on the ribosomes have been known for decades, genetic data on ribosomal mutations conferring resistance against Tc have been reported only recently. Ross et al. (11) 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 responsible for the inhibition of the A-site stable occupation. Recently Trieber et al. (12) have published a new set of data on mapping of Tc-resistant mutants in 16S rRNA which could be attributed (by us) to the Tet-4 site (10). We set out to collect data concerning the binding sites on the ribosomal 30S subunit of E.coli in solution. We have applied one of the widely used methods, photo-af®nity modi®cation, to map Tc-binding site(s) on the ribosomes. The Tc molecule has two uncoupled conjugated bond systems: ring A and rings B-C-D (Fig. 1A). The two ring systems are the reason for two peaks in the absorption spectrum of Tc (Fig. 1B). Irradiation of the Tc±ribosome complex with light of 365 nm excites the Tc molecule (13) and yields a covalent bond with reactive groups of the ribosome in the surrounding Tc (14). 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: kopylov@rnp-group.genebee.msu.su

Nucleic Acids Research, Vol. 32 No. 8 Ö Oxford University Press 2004; all rights reserved


Nucleic Acids Research, 2004, Vol. 32, No. 8

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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 subunits (no irradiation).

Figure 1. (A) Structure of Tc complex with Mg2+ (25). (B) Absorption spectrum of Tc.

E.coli. In addition to some nonspeci®cally modi®ed proteins: S18, S4, S14 and S13 (15), the protein S7 turned out to be the major target (16). Using a more advanced approach, Oehler et al. (17) also found a modi®cation of S7, as well as the 16S rRNA near the S7 binding site. Because both above mentioned groups of researchers had used a large molar excess of Tc over the ribosome, which could promote additional nonspeci®c binding [as was shown earlier for Tc binding to transcriptional repressor protein TetR(D) (14,18)], we paid particular attention to the Tc/ 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 modi®cations. MATERIALS AND METHODS Materials 30S ribosomal subunits of E.coli were isolated as described (19). 7-[3H]-Tc with a speci®c activity of 37 GBq/mmol was from New England Nuclear, USA. Photo-af®nity modi®cation For the complex formation, 30S subunits were pre-incubated for 10 min at 37°C in the buffer: 20 mM HEPES±KOH pH 7.6;

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 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 (DRSh-250, PhysPribor, Russia) has been used with the main emission maximum near 365 nm. Samples were irradiated for 2.5 min at 0°C, in a 313 nm cut-off plastic cuvette with 10 mm optical path (Sarstedt, Germany), which was positioned 25 cm away from the lamp. Primer extension analysis of the 16S rRNA modi®cations The 16S ribosome used for described rRNA was isolated from the irradiated Tc±30S complex by standard phenol extraction, and was reverse transcriptase primer extension analysis as (17,22).

RESULTS 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 been used. Our preliminary data on Tc interactions with E.coli 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 of Tc over the ribosome, but just proper concentrations of the components ([Tc] = 1 mM, [30S] = 2 mM), close to the


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Nucleic Acids Research, 2004, Vol. 32, No. 8 nucleotide was taken as the following nucleotide in the 16S rRNA template. 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. DISCUSSION Two 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 isolates (11,12), as well as some indirect data (5), also indicate that there could be at least one more binding site for Tc, though its location is not yet clear. The solution data, published earlier for E.coli ribosomes, as discussed in the Introduction, could not resolve this ambiguity, probably due to the use of a large excess of Tc over ribosomes in the experiments. In this study, we have revealed a second high af®nity Tc binding site within the 3¢-major domain of the 16S rRNA of the 30S subunit of E.coli ribosome, close to the Tet-4 site, in 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 to the Tc binding sites Tet-4 and Tet-6 (10). Our computer annotation of available X-ray data [PDB 1I97 (10)] has revealed the following picture (Fig. 3). Tc could modify C936 from either/both Tet-4 and Tet-6 binding sites, which are at an é equal distance of about 10 A from C936. On the other hand, both C936 and C948 could be modi®ed simultaneously, if Tet-4 was occupied as the only site. In this case, the distances é from Tet-4 to C936 and C948 are 9.8 and 14.2 A, respectively. The exact mechanism of Tc photolysis is not known in detail (13). Therefore, the probe±target distance for modi®cation with excited Tc molecules is not known either. The af®nity modi®cation event does not necessarily mean that reactive residues are in direct contact. For example, Lancaster et al. (24) have revealed that the distribution of probe±target distances for directed hydroxyl radical cleavages measured from the S8-16S rRNA models might be within the range of é 20 A, and even more. If one takes into account the size of the é Tc molecule of about 6 Q 12 A, the distances determined from the established Tc binding sites seem to be reasonable. In addition, Tc binding in solution with 30S subunits might

Figure 3. Putative sites of Tc interactions with the 16S rRNA within 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:// cn.expasy.org/spdbv). The 16S rRNA sequence numbering is according to E.coli. G942 corresponds to in vivo genetic data (12), C936 and C948 data from this publication. The ribosomal small subunit interface with six putative Tc binding sites is on the left. (A) The extracted structure of 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 sub-domain is the same as for the subunit. (B) Space-®lled Tet-4 and Tet-6 (black), and the 16S rRNA nucleotides (gray) are depicted with the same orientation as in (A). (C) The image is depicted at an orientation, different é from that in (B), to show the distances (A) in more detail.

corresponding value of the binding constant (2 Q 106 M±1) measured earlier (23). For photo-af®nity modi®cation, the Tc/30S subunit ratio was 1:2; 45% of the input Tc was bound to 30S subunits under this condition. The photo-af®nity reaction for the [3H]-Tc-30S subunit complex was triggered by irradiation at a wavelength of 365 nm, for 2.5 min at 0°C, which represents a short irradiation time compared with earlier studies (16). In addition, the buffer used contained mercaptoethanol to avoid light-independent incorporation of Tc photo-products (13,16). It turned out that the covalently linked [3H]-Tc-label was equally distributed between the 16S rRNA and the ribosomal proteins, as has been previously described (17). The 16S rRNA was isolated and analyzed by primer extension (17,22). The chosen set of primers allows scanning of the entire 16S rRNA sequence, except the very 3¢-end region. The 16S rRNAs both from 30S subunits irradiated without Tc and from non-irradiated 30S subunits were used as controls for identi®cation of random stops on the RNA template (Fig. 2, lines 2 and 3, respectively). When a stop was observed, the modi®ed


Nucleic Acids Research, 2004, Vol. 32, No. 8 induce subtle changes in this binding region, which could not be observed by binding to the rigid crystals. This would bring Tc even closer to the modi®ed nucleotides. In our previous publication (17), it was shown that a large excess of Tc could modify a different set of 16S rRNA nucleotides: G693, G1300 and G1338. There is no direct correlation between binding to the particular site and possible yield of cross-linking within the site. Therefore, if the excess of Tc modi®es more reactive nucleotides in some other sites, then the modi®cations described might have been masked. Our suggestion that the modi®cation could occur from the Tet-4 binding site perfectly correlates with recent in vivo genetic data for natural isolates of Tc-resistant strains of H.pylori or for arti®cially created strains, revealing that a deletion of G942 (helix H29) of the 16S rRNA confers moderate Tc resistance up to 8-fold (12). Figure 3 shows that é G942 is in very close proximity to Tet-4 (2.7 A). We can reconcile our observations in the following way. The ®rst binding site can be ascribed to the well accepted Asite related Tc binding site, Tet-1. And in a separate set of experiments on photo-af®nity modi®cation of the 30S subunit, we also revealed some ribosomal proteins in the vicinity of Tet-1 (M. M. Anokhina, Ts. A. Egorov, K. H. Nierhaus, B. Wittmann-Liebold, V. A. Spiridonova and A. M. Kopylov, manuscript in preparation). The second Tc binding site correlates well with the Tet-4 site. It remains to be determined whether Tc binding to the Tet-4 site contributes to the inhibition mechanism of this drug. ACKNOWLEDGEMENTS The paper is dedicated to the memory of Elena M. Kopylova. We thank C. Berens, E. Dobrov, V. Sergeyev, P. Sergiev, V. Ramakrishnan, T. Rassokhin; and special thanks to A. Bogdanov for permanent support and stimulating discussions. The work was supported by RFBR-OEAD 00±04±02007, RFBR 04±04±48942, and Universities of Russia UR-05.02.041. REFERENCES
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