| 1. |
- Cruz, Jose Almeida, et al.
(författare)
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RNA-Puzzles : A CASP-like evaluation of RNA three-dimensional structure prediction
- 2012
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 18:4, s. 610-625
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Tidskriftsartikel (refereegranskat)abstract
- We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.
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| 2. |
- Pettersson, Ethel, 1968-
(författare)
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Mechanism Studies of Catalytic RNA, and RNA and DNA Model Compounds
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes mechanism studies of transesterifications of phosphate esters. The aim of the studies concerns two fields. First, the catalytic mechanism that group I introns use in self-splicing. The focus was on metal ion coordinating groups at the catalytic site. Studies were performed on a (almost) full-length group I intron. A 2'-amino modification was introduced in the co-substrate and metal ion switch experiments were carried out. In the presence of magnesium ions the reaction rate decreased but introducing nitrogen preferring metal ions as for example manganese, this negative trend could be reversed. Further studies of the catalytic mechanism were performed on the same group I intron but converted to a ribozyme by deleting the substrate site. We introduced a 2'-amino group in the leaving group nucleoside, in a chemical synthesized substrate and metal ion switch experiments were carried out. At most we could study the cleavage reaction with two 2'-amino modifications at the catalytic site, in the co-substrate and in the substrate. In the presence of two 2'-amino modifications we obtained more than additive effects on the rate, in the presence of manganese ions. These experiments showed how one catalytic metal ion coordinated to the 2'-position in the co-substrate and a second one to the 2'-position in the leaving sugar nucleoside. An alternative model of the earlier proposed two metal ion mechanism used by group I introns, is presented.Five RNA model compounds, methyl furanosides with a 5-diphenyl phosphate group were synthesized. The metal ion promoted intramolecular cyclisation reaction, where the 3-hydroxyl attacks the 5-diphenyl phosphate was studied in the presence of different divalent metal ions. Two derivatives contained a 2-amino group and higher reaction rates were obtained in experiments with nitrogen preferring metal ions. These studies showed the effect of an adjacent position, on the hydroxy nucleophile, via coordination of a metal ion and also give data supporting the mechanism model for the catalytic RNA. The other field in this thesis concerns investigations on why ethylene oxide causes more DNA damages, than propylene oxide. To exclude that the intramolecular phosphate transesterification reaction was responsible for the observed differences, two dithymidine alkyl phosphates were synthesized and studied. Further, to see that the respective epoxide did not have different alkylation rates toward a phosphate center, the alkylation rate for the respective epoxide was studied. Neither of these events could be ascribed to be responsible for the earlier observations concerning ethylene oxide and propylene oxide.
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| 3. |
- Ren, Yan-Guo
(författare)
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Poly(A)-Specific Ribonuclease (PARN)
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Degradation of the mRNA 3'-end located poly(A) tail is an important step for mRNA decay in mammalian cells. Thus, to understand mRNA decay in detail, it is important to identify the catalytic activities involved in degrading poly(A). We identified and purified a 54-kDa polypeptide responsible for poly(A)-specific 3' exonuclease activity in calf thymus extracts. The 54-kDa polypeptide is a proteolytic fragment of the poly(A)-specific ribonuclease (PARN) 74-kDa polypeptide. PARN is a divalent metal ion dependent, poly(A)-specific, oligomeric, processive and cap interacting 3' exonuclease. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. The interaction with the 5'-end cap structure stimulates PARN activity and also amplifies the processivity of the deadenylation reaction. Furthermore, the cap binding site and the active site of PARN are separate from each other. To characterise the active site of PARN, we per-formed side-directed mutagenesis, Fe2+-mediated hydroxyl radical cleavage and metal ion switch experiments. We have demonstrated that the conserved acidic amino acid residues D28, E30, D292 and D382 of human PARN are essential for PARN activity and that these amino acid residues are directly involved in the co-ordination of at least two metal ions in the active site of PARN. Phosphorothioate modification on RNA substrates revealed that the pro-R oxygen atom of the scissile phosphate group interacts directly with the metal ion(s). Based on our studies, we propose a model for the action of PARN. Similarly to what has been observed for ribozymes, aminoglycoside antibiotics inhibit PARN activity, most likely by the displacement of catalytically important divalent metal ions. Among the aminoglycoside antibiotics tested, neomycin B is the most potent inhibitor. We speculate that inhibition of enzymes using similar catalytic mechanisms as PARN could be a reason for the toxic side effects caused by aminoglycoside antibiotics in clinical practice.
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