Crystallization and preliminary diffraction studies of the structural domain E of <i>Thermus flavus</i> 2S rRNA

Nolte, A; Kluβman, Sven; Lorenz, Siegfried; Bald, Rolf; Betzel, Christian; Dauter, Z.; Wilson, K.S.; Fürste, Jens P.; Erdmann, Volker A.

doi:10.1016/0014-5793(95)01136-3

Cited by 16 publications

(10 citation statements)

References 16 publications

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“…Domain E of 5S rRNA of Thermus flavus was prepared by solid phase chemical synthesis [6]and further purified by reversed phase HPLC. Crystals of the 20‐mer suitable for X‐ray analysis were obtained by vapor diffusion as reported before [9]. The space group was assigned to P3 2 21 with unit cell parameters of a = b =42.8 Å and c =162.2 Å.…”

Section: Methodsmentioning

confidence: 99%

“…The molecular structure of these parts of the 5S rRNA will be the first step to increase our knowledge of how proteins recognize and interact with ribosomal nucleic acids. We have already reported the crystal structure of domain A from Thermus flavus [7, 8]and preliminary diffraction studies of domain E [9]. Recently Correll et al [10]have presented the X‐ray structure of a 62 nt fragment of Escherichia coli 5S RNA mainly in helical conformation, which corresponds, according to Fig.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop

et al. 1998

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The synthetic RNA fragment 5P-CUGGGCGG(GC-GA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79^97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 A î and refined to an R-value of 24.1% also represents the first X-ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.z 1998 Federation of European Biochemical Societies.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop

et al. 1998

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“…Separation and Identification of Diastereomeric Oligoribonucleotides of identical sequence (13-mers), either unmodified or carrying a single phosphorothiote modification (5'-CCCUUUCsGCGGGA), were prepared by solid-phase synthesis essentially as described (5,6). Oligoribonucleotides were purified by reversed-phase HPLC on an ODS C18 Beckman Ultrasphere column at 45°C (5).…”

Section: Methodsmentioning

confidence: 99%

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

Warnecke

Fürste

Hardt

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

136

115

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To study the cleavage mechanism ofbacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp-or RNase P is an essential structure-specific endoribonuclease that generates the mature 5' ends of tRNAs. In vitro, RNA subunits of bacterial RNase P enzymes were shown to be catalytically active in the absence of the protein subunit (1). Processing of precursor tRNAs (ptRNAs) by RNase P is an essentially irreversible reaction yielding 3'-OH and 5'-phosphate termini. A solvent hydroxide is thought to act as the nucleophile in an SN2 in-line displacement mechanism (2, 3).To gain a deeper insight into the cleavage mechanism by Escherichia coli RNase P RNA, we have synthesized ptRNA substrates carrying a single Rp-or Sp-phosphorothioate modification at the RNase P cleavage site. The diastereomeric substrates were analyzed for gel-resolvable binding to RNase P RNA and were studied in single turnover experiments in the presence of different divalent metal ions, such as Mg2+, Mn2+, and Cd2+. The following results were obtained: (i) the Spdiastereomer moderately affected ptRNA ground state binding, while the Rp-diastereomer had no effect; (ii) cleavage by RNase P RNA involves direct metal ion coordination to the pro-Rp oxygen; (iii) there is no specific role for Mg2+ at the pro-Sp oxygen; and (iv) cleavage of the Rp-diastereomeric substrate has a lower cooperative dependence (nH = 1.8) upon[Cd2+] than cleavage of the unmodified substrate upon [Mg2+] (nH = 3.3). Implications for the unique RNase P RNA cleavage mechanism are discussed in the context of previously proposed mechanistic models (2-4). MATERIALS AND METHODS Separation and Identification of Diastereomeric 13-Mers.Oligoribonucleotides of identical sequence (13-mers), either unmodified or carrying a single phosphorothiote modification (5'-CCCUUUCsGCGGGA), were prepared by solid-phase synthesis essentially as described (5, 6). Oligoribonucleotides were purified by reversed-phase HPLC on an ODS C18 Beckman Ultrasphere column at 45°C (5). The material of the peak containing the two diastereomeric 13-mers was vacuumdried, and the Rp-and Sp-diastereomers were separated by a second reversed-phase run at 4°C. The assignment of configuration of the two diastereomeric 13-mers was accomplished by digestion with the stereoselective enzymes snake venom phosphodiesterase I or nuclease P1 essentially as described (6). Phosphorothioate-specific iodine hydrolysis was performed essentially as described recently (7).Assembly of the ptRNAGIY. Chemically synthesized RNA oligomers were phosphorylated at their 5' termini by T4 polynucleotide kinase; T7 transcripts were synthesized in the presence of excess 5'-GMP to obtain 5'-monophosphates (7). Modified or unmodified 13-mers, a second 11-nt RNA oligonucleotide (5'-GUAGCUCAGUC-3', obtained either by chemical RNA synthesis or T7 transcription), and the 3'-portion of the tRNA (obtained by T7 transcription, starting at G+ 18; see Fig. 1) were annealed to a bridging DNA oligonucleotide (complementary to...

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“…The E-domain of E. coli 5s rRNA (5'-rGUGUGGGGUCUCCC-CAUGC-3'), corresponding to nucleotides 79-97, was synthesized chemically [41].…”

Section: Unlahelled E-domain Of E Coli 5s Rrnamentioning

confidence: 99%

Initial analysis of 750 MHz NMR spectra of selectively ¹⁵N‐G,U labelled E. coli 5S rRNA

et al. 1996

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The overall folding of an RNA molecule is reflected in its base pairing pattern. The identification of that pattern provides a first step towards the determination of the structure of an RNA molecule. We show that the application of heteronuclear NMR methods at 750 MHz to E. coli 5S rRNA (120 nucleotides) selectively labelled with 15N in guanine and uridine allows observation of base pairing patterns for a larger RNA molecule. We also present evidence that the fold of the E-domain of the 5S rRNA (nt 79-97) as a contiguous part of the 5s rRNA and as an isolated molecule is virtually the same.

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Crystallization and preliminary diffraction studies of the structural domain E of Thermus flavus 2S rRNA

Cited by 16 publications

References 16 publications

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

Initial analysis of 750 MHz NMR spectra of selectively ¹⁵N‐G,U labelled E. coli 5S rRNA

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Crystallization and preliminary diffraction studies of the structural domain E of Thermus flavus 2S rRNA

Cited by 16 publications

References 16 publications

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

Initial analysis of 750 MHz NMR spectra of selectively 15N‐G,U labelled E. coli 5S rRNA

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Initial analysis of 750 MHz NMR spectra of selectively ¹⁵N‐G,U labelled E. coli 5S rRNA