Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution.

Arni, R.K.; Heinemann, Udo; Tokuoka, R.; Saenger, Wolfram

doi:10.1016/s0021-9258(19)37597-0

Cited by 159 publications

(36 citation statements)

References 54 publications

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“…Some theoretical studies predict that the ribose ring is extremely flexible and that the differences in energy between different ribose conformations are very small (Levitt & Warshel, 1978;Olson, 1982;Olson & Sussman, 1982), while others predict that the C2'-endo and C3'-endo conformations are much lower energy than other conformers (Murray-Rust & Motherwell, 1978;de Leeuw et al, 1980). Further support for the reasonableness of the calculated structures, however, is that their conformations are similar to those observed in different crystal structures of microbial ribonucleases complexed with nucleotides (Heinemann & Saenger, 1982;Arni et al, 1988;Koepke et al, 1989;Lenz et al, 1991b).…”

Section: Resultsmentioning

confidence: 75%

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Meiering¹,

Bycroft²,

Lubienski³

et al. 1993

Biochemistry

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The binding of 3'-GMP to the ribonuclease, barnase, has been studied using heteronuclear 2D and 3D NMR spectroscopy. The 1H and 15N NMR spectra of barnase complexed with 3'-GMP have been assigned. 2D and 3D NOESY spectra have been used to identify inter- and intramolecular NOEs, and a solution structure for the barnase-3'-GMP complex has been calculated. The position of the guanine ring of the ligand is reasonably well defined in the structures. The guanine ring forms hydrogen bonds with the NH protons of Ser57 and Arg59. These residues are located in a loop that is conserved among the microbial guanine-specific ribonucleases. The 2'-hydroxyl of 3'-GMP is close to His102 and Glu73, which have been shown to be involved in catalysis. The phosphate group of 3'-GMP is close to a number of positively charged residues that have also been shown to be important for activity. The position of the sugar moiety of 3'-GMP is less well defined in the structures. Structures calculated for the complex could not simultaneously satisfy all the observed intermolecular NOEs for the sugar protons, suggesting that the sugar samples several conformations when bound to barnase. The binding of 3'-GMP to barnase in solution is similar to that observed in the crystal structures of nucleotides bound to related ribonucleases. 3'-GMP binding causes no major conformational change in barnase. In contrast to the small structural changes that occur, there is a significant decrease in the rates of hydrogen/deuterium exchange and aromatic ring rotation in the active site of barnase upon ligand binding.

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Section: Resultsmentioning

confidence: 75%

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Meiering¹,

Bycroft²,

Lubienski³

et al. 1993

Biochemistry

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“…The crystal structure of RNase T 1 containing guanosine monophosphate in the binding site shows that the nucleobase adopts the syn conformation with various H-bonding contacts directing the corresponding recognition (Figure , G model built from information in refs and ). Three reasons led us to rationalize that 8-oxoG could be a substrate for RNase T 1 : (1) that 8-oxoG is known to exist in the syn conformation preferentially, (2) that the H-bonding sites present in G are still part of those in 8-oxoG, and (3) that the N1 and O6 positions, known to interact with the enzyme, are not modified in the 8-oxoG oxidative lesion.…”

Section: Discussionmentioning

confidence: 99%

Reactivity and Specificity of RNase T₁, RNase A, and RNase H toward Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine

et al. 2018

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Understanding how oxidatively damaged RNA interacts with ribonucleases is important because of its proposed role in the development and progression of disease. Thus, understanding structural aspects of RNA containing lesions generated under oxidative stress, as well as its interactions with other biopolymers, is fundamental. We explored the reactivity of RNase A, RNase T, and RNase H toward oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful for understanding (1) how these RNases can be used to characterize the structural impact that this lesion has on RNA and (2) how oxidatively modified RNA may be handled intracellularly. 8-OxoG was incorporated into 10-16-mers of RNA, and its reactivity with each ribonuclease was assessed via electrophoretic analyses, circular dichroism, and the use of other C8-purine-modified analogues (8-bromoguanosine, 8-methoxyguanosine, and 8-oxoadenosine). RNase T does not recognize sites containing 8-oxoG, while RNase A recognizes and cleaves RNA at positions containing this lesion while differentiating if it is involved in H-bonding. The selectivity of RNase A followed the order C > 8-oxoG ≈ U. In addition, isothermal titration calorimetry showed that an 8-oxoG-C3'-methylphosphate derivative can inhibit RNase A activity. Cleavage patterns obtained from RNase H displayed changes in reactivity in a sequence- and concentration-dependent manner and displayed recognition at sites containing the modification in some cases. These data will aid in understanding how this modification affects reactivity with ribonucleases and will enable the characterization of global and local structural changes in oxidatively damaged RNA.

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“…In addition to the main difference at the vault caps, there is also a subtle change in the weak internal density of the intact and RNase-treated vault recon-structions+ As previously reported, density above the background noise level can be seen in the central slice of the intact vault reconstruction (Fig+ 6A)+ The equivalent central slice of the RNase-treated vault reconstruction reveals less density in the vault cavity (Fig+ 6B)+ Admittedly, different symmetries, C8 versus D8, were imposed on these two reconstruc-tions+ However, the C8 intact vault reconstruction has similar internal weak density in both halves of the vault, so the contents would probably not be too different with imposed D8 symmetry+ The cryo-EM particle images of the RNase-treated vaults show that there is still significant density trapped within the vault (Fig+ 2); therefore RNase treatment has not completely degraded the contents, but it might have partially degraded them+ Comparison of the intact and RNase-treated vault reconstructions raises the possibility that some portion of the contents of the intact vault is RNA, either vRNA or another RNA species+ The molecular dimensions observed in the crystal structure of RNase T1 (Arni et al+, 1988) suggest that this enzyme might fit through the small holes observed in the reconstruction (;20 ϫ 40 Å)+ Perhaps the smaller RNase A is able to pass through more easily+ The size of the holes in the vault reconstruction is dependent on the isosurface value chosen, and at higher isosurface values, the holes appear larger+ We have set the isosurface value of the RNase-treated reconstruction so that the volume corresponds with the average STEM mass measurement of the vault minus 5% for the vRNA+ Also, we suspect that the caps do not follow perfect dihedral eightfold symmetry, and portions of the true cap structure may be blurred in the reconstruction+ In support of this idea, we note that only the flat ends of the caps show strong density in the reconstruction, and the side walls of the caps appear weak as if they have been inappropriately averaged (Fig+ 6B)+ So perhaps there are larger openings in the caps that are not observed in the symmetrized reconstruction, but that would allow both types of RNase molecules to enter the vault cavity+ FIGURE 5. Difference imaging between the RNase-treated and intact vault reconstructions to reveal the location of the vRNA+ The difference density above the noise level is shown in red, overlaid on two views of the intact vault reconstruction that has been cropped in half+ Note that the difference density is located at the ends of the vault caps+ The scale bar corresponds to 250 Å+ FIGURE 6.…”

Section: Internal Contents Of the Vaultmentioning

confidence: 99%

RNA location and modeling of a WD40 repeat domain within the vault

Kong

Siva

Kickhoefer

et al. 2000

RNA

133

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The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.

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Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution.

Cited by 159 publications

References 54 publications

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Reactivity and Specificity of RNase T₁, RNase A, and RNase H toward Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine

RNA location and modeling of a WD40 repeat domain within the vault

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Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution.

Cited by 159 publications

References 54 publications

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy

Reactivity and Specificity of RNase T1, RNase A, and RNase H toward Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine

RNA location and modeling of a WD40 repeat domain within the vault

Contact Info

Product

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About

Reactivity and Specificity of RNase T₁, RNase A, and RNase H toward Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydroguanosine