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A protonated base pair participating in rRNA tertiary structural interactions

A protonated base pair participating in rRNA tertiary structural interactions © 2001 Oxford University Press Nucleic Acids Research, 2001, Vol. 29, No. 24 5067–5070 A protonated base pair participating in rRNA tertiary structural interactions Andriy V. Kubarenko, Petr V. Sergiev, Alexey A. Bogdanov, Richard Brimacombe and Olga A. Dontsova* Department of Chemistry, Moscow State University, Moscow 119899, Russia and Max-Planck-Institut fur Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Received August 14, 2001; Revised and Accepted October 22, 2001 ABSTRACT structure(1).Furthermore,wehavemadeuse of theatomic structures (1,2) in order to evaluate the reliability of the data In the recently published X-ray crystallographic obtained for the E.coli ribosome from the various crosslinking structure for the 50S subunit of Haloarcula and other chemical approaches which were exploited in the marismortui ribosomes, residue U2546 of the 23S rRNA model-building studies (7). forms a non-Watson–Crick base pair with U2610. The Here we extend our correlation of the E.coli and H.marismortui corresponding residues in the secondary structure structures to a more detailed level and describe the detection of of the Escherichia coli 23S molecule are U2511 and a non-Watson–Crick CH -U base pair in E.coli that is isostruc- tural to a U-U pair in H.marismortui. The existence of this pair C2575, and it follows that the latter base (C2575) was at first predicted from a comparison of the respective should be protonated in order to form a base pair that secondary structures of the 23S rRNA molecules with the is isostructural with its counterpart in H.marismortui. atomic structure for H.marismortui. Experimental proof was This prediction was demonstrated experimentally by then obtained by reduction of the protonated base by treatment reduction with sodium borohydride followed by with sodium borohydride, followed by primer extension analysis primer extension analysis; borohydride is able to to identify the locations of the reduction products. In addition reduce positively charged bases, yielding products + to the CH -U pair, a complete scan of the borohydride-treated which block reverse transcription. In the course of the 16S and 23S rRNAs by primer extension revealed an AH -G analysis a further charged base pair (AH 1528-G1543) protonated base pair in another region of the 23S rRNA. This technique should be generally applicable for the identification of was identified in the E.coli 23S molecule. Both + + base pairs containing protonated adenine or cytidine residues in charged pairs (U2511-CH 2575 and AH 1528-G1543) any RNA or RNP. were only observed in the context of the intact ribo- somal subunit and were not seen in deproteinized rRNA. MATERIALS AND METHODS Ultrapure rNTPs and dNTPs were obtained from Pharmacia, alkaline phosphatase, T4 polynucletide kinase and reverse INTRODUCTION transcriptase from Boehringer Mannheim, [γ- P]ATP from Atomic structures for both the large and small ribosomal subunits Amersham, sodium borohydride from Sigma. have recently been determined by X-ray crystallography, that of Sodium borohydride treatment the 50S subunit using ribosomes from Haloarcula marismortui (1) and that of the 30S subunit using ribosomes from Thermus 23S and 16S rRNA were treated with sodium borohydride in thermophilus (2,3). On the other hand, the vast majority of the both 70S ribosomes and in the deproteinized state in solution. biochemical data relating to ribosomal structure has been For this purpose an aqueous solution of sodium borohydride obtained with ribosomes from Escherichia coli. For this (15 µ l, 3.3 mg/ml) was added to 60 pmol 23S plus 16S rRNA organism, the best structural information so far available has or 70S ribosomes in 135 µ l of buffer (50 mM Tris–HCl pH 8.0, been obtained by cryo-electron microscopy and, prior to the 70 mM NH Cl, 30 mM KCl, 7 mM MgCl , 1 mM DTT). The 4 2 advent of X-ray crystallographic structures, computer models mixture was kept on ice for 30 min in the dark and the reaction for the E.coli 16S and 23S rRNA were derived by combining was then stopped by addition of 15 µ l of 3 M sodium acetate the biochemical data with these cryo-electron microscopic pH 5.5, and 450 µ l of ethanol. For the ribosomal samples, after structures (4,5). As part of a program to correlate the structures precipitation the pellets were dissolved in 50 µ lof buffer of the E.coli ribosomal subunits with those of the H.marismortui (0.3 M NaOAc pH 7.0, 0.5% SDS, 5 mM EDTA) and rRNA and T.thermophilus subunits, we have recently compared (6) was purified by phenol treatment (twice with 50 µ l of phenol the locations of several helices in the model for the E.coli 50S and once with 50 µ l of chloroform) and precipitated with subunit (5) with their corresponding locations in the atomic ethanol for 2 h at –20°C. For the 16S/23S rRNA samples this *To whom correspondence should be addressed. Tel: +7 095 939 5418; Fax: +7 095 939 3181; Email: dontsova@genebee.msu.su Correspondence may also be addressed to Petr V. Sergiev. Tel: +7 095 939 5418; Fax: +7 095 939 3181; Email: petya@genebee.msu.su 5068 Nucleic Acids Research, 2001, Vol. 29, No. 24 Figure 1. The base pair U2610-U2546 in H.marismortui and its location in the 3D structure of the 50S subunit. The view of the 50S subunit is from the inter- face side and the positions of the peptidyltransferase center (PTC), central protuberance (CP) and L1 and L7/L12 proteins are indicated. Figure 2. Structures of base pairs and reduction products. (A) Structures of the U2610-U2546 base pair in H.marismortui and of the isostructural C2575-U2511 phenol treatment was carried out prior to the reaction with pair in E.coli.(B) Protonated bases and their products of reduction by borohydride. sodium borohydride. From top to bottom, N1-methyladenine, adenine, cytosine. (C) Structure of the G-AH base pair. Primer extension Full-length screening of the 23S rRNA was made using 12 primers complementary to nt 310–330, 559–568, 765–785, quaternary structure of the peptidyltransferase region of the 1040–1059, 1320–1339, 1619–1636, 1831–1850, 2081–2100, 23S rRNA. However, for the C-U pair in E.coli to be isostruc- 2281–2301, 2487–2503, 2730–2749 and 2886–2903, that of tural with the U-U pair in H.marismortui the cytosine moiety the 16S rRNA with eight primers complementary to nt 162–178, would have to be protonated at position N3 (Fig. 2A). In fact, a 324–340, 481–497, 684–700, 838–854, 1047–1063, 1310–1326 protonated CH -U pair with exactly this geometry was recently and 1457–1473. The primer extension reaction was carried out discovered by Blanchard and Puglisi in the structure of an as described (8). oligonucleotide analog of the ‘A loop’ region of E.coli 23S rRNA (10); the pair existed at pH 5.5 but was rearranged at higher pH (pH 7.5). It should be noted that the structure of this RESULTS AND DISCUSSION CH -U pair (Fig. 2A) is radically different from the single In the atomic structure of H.marismortui 23S rRNA (1) there is hydrogen bonded C-U pair found in the self-complementary a U-U base pair between residues U2610 and U2546 in helix dodecamer duplex (GGACUUCGGUCC) (11) or that in the 90, which is located close to the peptidyltransferase region bifurcated hydrogen-bonded C-U pair described by Auffinger (Fig. 1). This pair has the same structure as that reported (9) for and Westhof (12). the universally conserved U-U pair in the A site region of the Sodium borohydride was chosen as a reagent to probe for the rRNA from the small subunit. A search through the known existence of such protonated residues by analogy with its secondary structures of the large subunit rRNAs of Archaea, ability to reduce N1-methylated adenine (Fig. 2B) (13). As a Eubacteria and Eucaria reveals that in the Archaea and result of the methylation the adenine residue carries a positive Eucaria the U residues corresponding to U2610 and U2546 in charge, which is distributed between the N1 and N6 nitrogen H.marismortui are both highly conserved. On the other hand, atoms. When this substrate is reduced with sodium borohydride in the Eubacteria the upstream U is highly conserved but the the six-membered ring loses its planar conformation and downstream residue is a C; in E.coli these residues are U2511 aromatic character. Both protonated adenine (unmethylated) and and C2575, respectively. From the level of conservation and its location in the 50S subunit, it seems likely that this base pair cytidine should have a similar structure to that of N-methylated could be important for maintenance of the tertiary and adenine; in the case of cytidine the positive charge is distributed Nucleic Acids Research, 2001, Vol. 29, No. 24 5069 structures containing CH in d(C )(15)orinatriplexsuchas CH GC (2,16,17) can exist at pH values close to 7. The primer extension scan of the 23S rRNA revealed a second minor site of reaction with sodium borohydride at posi- tion A1528 in helix 59 of the E.coli 23S rRNA (Fig. 3B). As with CH 2575, this modification was only observed when the borohydride reduction was made with 70S ribosomes, but not with free rRNA (lanes 3 and 1, respectively). The phylogeny of A1528 is somewhat more complex than that of C2575, because in the Archaea and in some Eucaria helix 59 is absent. However, in many Eucaria there is an A-U pair between the residues that correspond to A1528 and G1543 in E.coli.In the Eubacteria the respective residues are either A and G (as in E.coli) or occasionally A and A, although in some cases helix 59 is missing here too. Taken together with the borohydride data of Figure 3B, this suggests that in E.coli there is an AH 1528-G1543 base pair, and indeed such a pair (Fig. 2C) has recently been reported (18), which is essentially isostruc- tural to a normal A-U Watson–Crick pair. Since helix 59 is absent in H.marismortui it is difficult to draw any conclusions with regard to the importance of this pair for maintenance of the ribosomal quaternary structure. Furthermore, the weakness of the primer extension signal (Fig. 3B) suggests that the A1528 residue may not be fully protonated and hence only partially reduced by the borohydride treatment. It has been shown (19) that the peptidyltransferase center of E.coli contains an adenine residue (A2451) with an unusually high pK value of ∼7.5, which has been proposed to be directly Figure 3. Primer extension analyses of 23S rRNA after sodium borohydride involved in the catalysis of peptide bond formation. In our treatment. (A) The area around nucleotide C2575. The sequencing lanes are experiments we did not find this residue to be protonated, but, marked A, C, G and U, respectively. Lanes 1 and 2 are from an experiment with since we carried out the borohydride reduction at pH 8.0, there deproteinized 23S rRNA in solution, lane 1 with borohydride treatment, lane 2 without. Lanes 3, 3′,4 and 4′ are from two independent experiments with 70S is no inconsistency here. We suggest that the method we have ribosomes, lanes 3 and 3′ with borohydride treatment, lanes 4 and 4′ without. described in this paper should prove useful as a direct and (B) The area around nucleotide A1528. The sequencing lanes are marked as in sensitive technique for the detection of protonated base pairs in (A). Lanes 1 and 2 are from an experiment with deproteinized 23S rRNA, with any RNA molecule or RNP complex. and without borohydride treatment, respectively, and lanes 3 and 4 from an experiment with 70S ribosomes, again with and without borohydride treat- ment. ACKNOWLEDGEMENTS This work was supported by grants from the Russian Foundation between the N3 and N4 nitrogen atoms (Fig. 2B). In the for Basic Research (99-04-49054), the Volkswagen-Stiftung reduced form these residues should be unable to form normal (I/74598) and the Howard Hughes Medical Institute Watson–Crick base pairs and, as a consequence, should be (HHMI55000303). A.B. acknowledges support from the Alex- detectable by primer extension analysis. ander von Humboldt Foundation. Accordingly, we made a reverse transcriptase scan of the complete 16S and 23S rRNA molecules from E.coli,after REFERENCES sodium borohydride reduction of either 70S ribosomes or isolated 16S/23S rRNA (see Materials and Methods). As can 1. Ban,N., Nissen,P., Hansen,J., Moore,P.B. and Steitz,T.A. (2000) be seen from Figure 3A, there is a strong reverse transcriptase The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science, 289, 905–920. stop at position 2576 after borohydride treatment of 23S rRNA 2. Wimberly,B.T., Brodersen,D.E., Clemons,W.M.,Jr, Morgan-Warren,R.J., in 70S ribosomes (lanes 3 and 3′), indicating that residue Carter,A.P., Vonrhein,C., Hartsch,T. and Ramakrishnan,V. (2000) C2575 was indeed modified. In contrast, no corresponding Structure of the 30S ribosomal subunit. Nature, 407, 327–339. signal was observed when the borohydride treatment was 3. Tocilj,A., Schlunzen,F., Janell,D., Gluhmann,M., Hansen,H.A., Harms,J., carried out using isolated rRNA (Fig. 3A, lane 1). Thus, the Bashan,A., Bartels,H., Agmon,I., Franceschi,F. and Yonath,A. (1999) The small ribosomal subunit from Thermus thermophilus at 4.5 Å resolution: CH 2575-U2511 base pair would appear to be stabilized in the pattern fittings and the identification of a functional site. Proc. Natl Acad. ribosomal structure by RNA–RNA or RNA–protein interac- Sci. USA, 96, 14252–14257. tions and this stabilization does not occur with isolated rRNA. 4. Mueller,F. and Brimacombe,R. (1997) A new model for the three- dimensional folding of Escherichia coli 16S ribosomal RNA. I. Fitting the The apparent pK of protonated cytidine in the nucleoside is + RNA to a 3D electron microscopic map at 20Å. J. Mol. Biol., 271, 4.3 (14). Although a CH -U base pair involving protonated 524–544. cytosine has only recently been observed (1,9), its existence is 5. Mueller,F., Sommer,I., Baranov,P., Matadeen,R., Stoldt,M., Wohnert,J., not surprising, since it is well known that the involvement of Gorlach,M., van Heel,M. and Brimacombe,R. (2000) The 3D arrangement cytosine in base pairing strongly increases its pK ; for example, of the23S and5SrRNA inthe Escherichia coli 50S ribosomal subunit a 5070 Nucleic Acids Research, 2001, Vol. 29, No. 24 based on a cryo-electron microscopic reconstruction at 7.5 Å resolution. 12. Auffinger,P. and Westhof,E. (1999) Single and bifurcated hydrogen- J. Mol. Biol., 298, 35–59. bonded base-pair in tRNA anticodon hairpins and ribozymes. 6. Matadeen,R., Sergiev,P., Leonov,A., Pape,T., van der Sluis,E., J. Mol. Biol., 292, 467–483. Mueller,F., Osswald,M., von Knoblauch,K., Brimacombe,R., 13. Macon,J.B. and Wolfenden,R. (1968) 1-Methyladenosine. Dimroth Bogdanov,A., van Heel,M. and Dontsova,O. (2001) Direct localization by rearrangement and reversible reduction. Biochemistry, 7, 3453–3458. cryo-electron microscopy of secondary structural elements in 14. Hartman,K.A.,Jr and Rich,A. (1965) The tautomeric form of helical Escherichia coli 23 S rRNA which differ from the corresponding regions polyribocytidylic acid. J. Am. Chem. Soc., 87, 2033–2039. in Haloarcula marismortui. J. Mol. Biol., 307, 1341–1349. 15. Chen,L., Cai,L., Zhang,X. and Rich,A. (1994) Crystal structure of a 7. Sergiev,P.V., Dontsova,O.A. and Bogdanov,A.A. (2001) Biochemical four-stranded intercalated DNA: d(C4). Biochemistry, 33, 13540–13546. methods for the structural study of prokaryotic ribosome: judgment day. 16. Holland,J.A. and Hoffman,D.W. (1996) Structural features and stability Mol. Biol. (Mosk.), 35, 1–25. 8. Rinke-Appel,J., Junke,N., Stade,K. and Brimacombe,R. (1991) The path of an RNA triple helix in solution. Nucleic Acids Res., 24, 2841–2848. of mRNA through the Escherichia coli ribosome; site-directed 17. Brodsky,A.S., Erlacher,H.A. and Williamson,J.R. (1998) NMR evidence cross-linking of mRNA analogues carrying a photo-reactive label at for a base triple in the HIV-2 TAR C-G.C+ mutant–argininamide various points 3′ to the decoding site. EMBO J., 10, 2195–2202. complex. Nucleic Acids Res., 26, 1991–1995. 9. Lynch,S.R. and Puglisi,J.D. (2001) Structure of eukaryotic decoding 18. Pan,B., Mitra,S.N. and Sundaralingam,M. (1999) Crystal structure of an region A-site RNA. J. Mol. Biol., 306, 1023–1035. RNA 16-mer duplex R(GCAGAGUUAAAUCUGC) with nonadjacent 10. Blanchard,S.C. and Puglisi,J.D. (2001) Solution structure of the A loop of G(syn).A+(anti) mispairs. Biochemistry, 38, 2826–2831. 23S ribosomal RNA. Proc. Natl Acad. Sci. USA, 98, 3720–3725. 19. Muth,G.W., Ortoleva-Donnelly,L. and Strobel,S.A. (2000) A single 11. Holbrook,S.H., Cheong,C., Tinoco,I.,Jr and Kim,S.-H. (1991) Crystal adenosine with a neutral pK in the ribosomal peptidyl transferase center. structure of a RNA double helix incorporating a track of non Watson- Crick base pairs. Nature, 353, 579–581. Science, 289, 947–950. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

A protonated base pair participating in rRNA tertiary structural interactions

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10.1093/nar/29.24.5067
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© 2001 Oxford University Press Nucleic Acids Research, 2001, Vol. 29, No. 24 5067–5070 A protonated base pair participating in rRNA tertiary structural interactions Andriy V. Kubarenko, Petr V. Sergiev, Alexey A. Bogdanov, Richard Brimacombe and Olga A. Dontsova* Department of Chemistry, Moscow State University, Moscow 119899, Russia and Max-Planck-Institut fur Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Received August 14, 2001; Revised and Accepted October 22, 2001 ABSTRACT structure(1).Furthermore,wehavemadeuse of theatomic structures (1,2) in order to evaluate the reliability of the data In the recently published X-ray crystallographic obtained for the E.coli ribosome from the various crosslinking structure for the 50S subunit of Haloarcula and other chemical approaches which were exploited in the marismortui ribosomes, residue U2546 of the 23S rRNA model-building studies (7). forms a non-Watson–Crick base pair with U2610. The Here we extend our correlation of the E.coli and H.marismortui corresponding residues in the secondary structure structures to a more detailed level and describe the detection of of the Escherichia coli 23S molecule are U2511 and a non-Watson–Crick CH -U base pair in E.coli that is isostruc- tural to a U-U pair in H.marismortui. The existence of this pair C2575, and it follows that the latter base (C2575) was at first predicted from a comparison of the respective should be protonated in order to form a base pair that secondary structures of the 23S rRNA molecules with the is isostructural with its counterpart in H.marismortui. atomic structure for H.marismortui. Experimental proof was This prediction was demonstrated experimentally by then obtained by reduction of the protonated base by treatment reduction with sodium borohydride followed by with sodium borohydride, followed by primer extension analysis primer extension analysis; borohydride is able to to identify the locations of the reduction products. In addition reduce positively charged bases, yielding products + to the CH -U pair, a complete scan of the borohydride-treated which block reverse transcription. In the course of the 16S and 23S rRNAs by primer extension revealed an AH -G analysis a further charged base pair (AH 1528-G1543) protonated base pair in another region of the 23S rRNA. This technique should be generally applicable for the identification of was identified in the E.coli 23S molecule. Both + + base pairs containing protonated adenine or cytidine residues in charged pairs (U2511-CH 2575 and AH 1528-G1543) any RNA or RNP. were only observed in the context of the intact ribo- somal subunit and were not seen in deproteinized rRNA. MATERIALS AND METHODS Ultrapure rNTPs and dNTPs were obtained from Pharmacia, alkaline phosphatase, T4 polynucletide kinase and reverse INTRODUCTION transcriptase from Boehringer Mannheim, [γ- P]ATP from Atomic structures for both the large and small ribosomal subunits Amersham, sodium borohydride from Sigma. have recently been determined by X-ray crystallography, that of Sodium borohydride treatment the 50S subunit using ribosomes from Haloarcula marismortui (1) and that of the 30S subunit using ribosomes from Thermus 23S and 16S rRNA were treated with sodium borohydride in thermophilus (2,3). On the other hand, the vast majority of the both 70S ribosomes and in the deproteinized state in solution. biochemical data relating to ribosomal structure has been For this purpose an aqueous solution of sodium borohydride obtained with ribosomes from Escherichia coli. For this (15 µ l, 3.3 mg/ml) was added to 60 pmol 23S plus 16S rRNA organism, the best structural information so far available has or 70S ribosomes in 135 µ l of buffer (50 mM Tris–HCl pH 8.0, been obtained by cryo-electron microscopy and, prior to the 70 mM NH Cl, 30 mM KCl, 7 mM MgCl , 1 mM DTT). The 4 2 advent of X-ray crystallographic structures, computer models mixture was kept on ice for 30 min in the dark and the reaction for the E.coli 16S and 23S rRNA were derived by combining was then stopped by addition of 15 µ l of 3 M sodium acetate the biochemical data with these cryo-electron microscopic pH 5.5, and 450 µ l of ethanol. For the ribosomal samples, after structures (4,5). As part of a program to correlate the structures precipitation the pellets were dissolved in 50 µ lof buffer of the E.coli ribosomal subunits with those of the H.marismortui (0.3 M NaOAc pH 7.0, 0.5% SDS, 5 mM EDTA) and rRNA and T.thermophilus subunits, we have recently compared (6) was purified by phenol treatment (twice with 50 µ l of phenol the locations of several helices in the model for the E.coli 50S and once with 50 µ l of chloroform) and precipitated with subunit (5) with their corresponding locations in the atomic ethanol for 2 h at –20°C. For the 16S/23S rRNA samples this *To whom correspondence should be addressed. Tel: +7 095 939 5418; Fax: +7 095 939 3181; Email: dontsova@genebee.msu.su Correspondence may also be addressed to Petr V. Sergiev. Tel: +7 095 939 5418; Fax: +7 095 939 3181; Email: petya@genebee.msu.su 5068 Nucleic Acids Research, 2001, Vol. 29, No. 24 Figure 1. The base pair U2610-U2546 in H.marismortui and its location in the 3D structure of the 50S subunit. The view of the 50S subunit is from the inter- face side and the positions of the peptidyltransferase center (PTC), central protuberance (CP) and L1 and L7/L12 proteins are indicated. Figure 2. Structures of base pairs and reduction products. (A) Structures of the U2610-U2546 base pair in H.marismortui and of the isostructural C2575-U2511 phenol treatment was carried out prior to the reaction with pair in E.coli.(B) Protonated bases and their products of reduction by borohydride. sodium borohydride. From top to bottom, N1-methyladenine, adenine, cytosine. (C) Structure of the G-AH base pair. Primer extension Full-length screening of the 23S rRNA was made using 12 primers complementary to nt 310–330, 559–568, 765–785, quaternary structure of the peptidyltransferase region of the 1040–1059, 1320–1339, 1619–1636, 1831–1850, 2081–2100, 23S rRNA. However, for the C-U pair in E.coli to be isostruc- 2281–2301, 2487–2503, 2730–2749 and 2886–2903, that of tural with the U-U pair in H.marismortui the cytosine moiety the 16S rRNA with eight primers complementary to nt 162–178, would have to be protonated at position N3 (Fig. 2A). In fact, a 324–340, 481–497, 684–700, 838–854, 1047–1063, 1310–1326 protonated CH -U pair with exactly this geometry was recently and 1457–1473. The primer extension reaction was carried out discovered by Blanchard and Puglisi in the structure of an as described (8). oligonucleotide analog of the ‘A loop’ region of E.coli 23S rRNA (10); the pair existed at pH 5.5 but was rearranged at higher pH (pH 7.5). It should be noted that the structure of this RESULTS AND DISCUSSION CH -U pair (Fig. 2A) is radically different from the single In the atomic structure of H.marismortui 23S rRNA (1) there is hydrogen bonded C-U pair found in the self-complementary a U-U base pair between residues U2610 and U2546 in helix dodecamer duplex (GGACUUCGGUCC) (11) or that in the 90, which is located close to the peptidyltransferase region bifurcated hydrogen-bonded C-U pair described by Auffinger (Fig. 1). This pair has the same structure as that reported (9) for and Westhof (12). the universally conserved U-U pair in the A site region of the Sodium borohydride was chosen as a reagent to probe for the rRNA from the small subunit. A search through the known existence of such protonated residues by analogy with its secondary structures of the large subunit rRNAs of Archaea, ability to reduce N1-methylated adenine (Fig. 2B) (13). As a Eubacteria and Eucaria reveals that in the Archaea and result of the methylation the adenine residue carries a positive Eucaria the U residues corresponding to U2610 and U2546 in charge, which is distributed between the N1 and N6 nitrogen H.marismortui are both highly conserved. On the other hand, atoms. When this substrate is reduced with sodium borohydride in the Eubacteria the upstream U is highly conserved but the the six-membered ring loses its planar conformation and downstream residue is a C; in E.coli these residues are U2511 aromatic character. Both protonated adenine (unmethylated) and and C2575, respectively. From the level of conservation and its location in the 50S subunit, it seems likely that this base pair cytidine should have a similar structure to that of N-methylated could be important for maintenance of the tertiary and adenine; in the case of cytidine the positive charge is distributed Nucleic Acids Research, 2001, Vol. 29, No. 24 5069 structures containing CH in d(C )(15)orinatriplexsuchas CH GC (2,16,17) can exist at pH values close to 7. The primer extension scan of the 23S rRNA revealed a second minor site of reaction with sodium borohydride at posi- tion A1528 in helix 59 of the E.coli 23S rRNA (Fig. 3B). As with CH 2575, this modification was only observed when the borohydride reduction was made with 70S ribosomes, but not with free rRNA (lanes 3 and 1, respectively). The phylogeny of A1528 is somewhat more complex than that of C2575, because in the Archaea and in some Eucaria helix 59 is absent. However, in many Eucaria there is an A-U pair between the residues that correspond to A1528 and G1543 in E.coli.In the Eubacteria the respective residues are either A and G (as in E.coli) or occasionally A and A, although in some cases helix 59 is missing here too. Taken together with the borohydride data of Figure 3B, this suggests that in E.coli there is an AH 1528-G1543 base pair, and indeed such a pair (Fig. 2C) has recently been reported (18), which is essentially isostruc- tural to a normal A-U Watson–Crick pair. Since helix 59 is absent in H.marismortui it is difficult to draw any conclusions with regard to the importance of this pair for maintenance of the ribosomal quaternary structure. Furthermore, the weakness of the primer extension signal (Fig. 3B) suggests that the A1528 residue may not be fully protonated and hence only partially reduced by the borohydride treatment. It has been shown (19) that the peptidyltransferase center of E.coli contains an adenine residue (A2451) with an unusually high pK value of ∼7.5, which has been proposed to be directly Figure 3. Primer extension analyses of 23S rRNA after sodium borohydride involved in the catalysis of peptide bond formation. In our treatment. (A) The area around nucleotide C2575. The sequencing lanes are experiments we did not find this residue to be protonated, but, marked A, C, G and U, respectively. Lanes 1 and 2 are from an experiment with since we carried out the borohydride reduction at pH 8.0, there deproteinized 23S rRNA in solution, lane 1 with borohydride treatment, lane 2 without. Lanes 3, 3′,4 and 4′ are from two independent experiments with 70S is no inconsistency here. We suggest that the method we have ribosomes, lanes 3 and 3′ with borohydride treatment, lanes 4 and 4′ without. described in this paper should prove useful as a direct and (B) The area around nucleotide A1528. The sequencing lanes are marked as in sensitive technique for the detection of protonated base pairs in (A). Lanes 1 and 2 are from an experiment with deproteinized 23S rRNA, with any RNA molecule or RNP complex. and without borohydride treatment, respectively, and lanes 3 and 4 from an experiment with 70S ribosomes, again with and without borohydride treat- ment. ACKNOWLEDGEMENTS This work was supported by grants from the Russian Foundation between the N3 and N4 nitrogen atoms (Fig. 2B). In the for Basic Research (99-04-49054), the Volkswagen-Stiftung reduced form these residues should be unable to form normal (I/74598) and the Howard Hughes Medical Institute Watson–Crick base pairs and, as a consequence, should be (HHMI55000303). A.B. acknowledges support from the Alex- detectable by primer extension analysis. ander von Humboldt Foundation. 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Nucleic Acids ResearchOxford University Press

Published: Dec 15, 2001

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