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Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse.

Ganichkin OM, Anedchenko EA, Wahl MC - PLoS ONE (2011)

Bottom Line: Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop.Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome.Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Strukturbiochemie, Freie Universität Berlin, Berlin, Germany.

ABSTRACT

Background: Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood.

Methodology/principal findings: We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec).

Conclusions/significance: We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins.

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Non-denaturing RNA purification.(A) Elution profile of in vitro transcribed mouse tRNASec from a MonoQ column. Peak 1 – unincorporated rNTPs, T7 RNA polymerase and other proteins; Peak 2 – abortive synthesis transcripts; Peak 3 – desired RNA sample; Peak 4 – aggregates or higher molecular weight nucleic acids. The gradient (buffer B from 30 to 100%) is shown as a dashed line. (B) Denaturing SDS PAGE analysis of peak fractions from Peaks 1–3. T7 RNA polymerase and molecular weight markers (M) were loaded as references. Protein bands were stained with Coomassie. (C) Denaturing urea PAGE analysis of peak fractions eluted from the MonoQ column. S – crude transcription extract. RNA bands were stained with methylene blue. (D) Elution profile of mouse tRNASec from a Superdex 75 10/300 GL column.
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pone-0020032-g001: Non-denaturing RNA purification.(A) Elution profile of in vitro transcribed mouse tRNASec from a MonoQ column. Peak 1 – unincorporated rNTPs, T7 RNA polymerase and other proteins; Peak 2 – abortive synthesis transcripts; Peak 3 – desired RNA sample; Peak 4 – aggregates or higher molecular weight nucleic acids. The gradient (buffer B from 30 to 100%) is shown as a dashed line. (B) Denaturing SDS PAGE analysis of peak fractions from Peaks 1–3. T7 RNA polymerase and molecular weight markers (M) were loaded as references. Protein bands were stained with Coomassie. (C) Denaturing urea PAGE analysis of peak fractions eluted from the MonoQ column. S – crude transcription extract. RNA bands were stained with methylene blue. (D) Elution profile of mouse tRNASec from a Superdex 75 10/300 GL column.

Mentions: We PCR-assembled DNA fragments encompassing a T7 RNA polymerase promoter and the RNA coding region and inserted the constructs into plasmids. The plasmids served as templates for amplification of the promoter and insert regions, using reverse primers with two 2′-O-methyl modified nucleotides at their 5′-ends to avoid 3′-end heterogeneity of the RNA products in the subsequent transcription reactions [28], [29]. The modified PCR products served as templates for in vitro transcription by T7 RNA polymerase. After removal of the DNA template by incubation with RNase-free DNase, the sample was directly loaded on a strong anion exchange column. None of the proteins present in the reaction mixture bound to the column under conditions, which afforded efficient binding and separation of RNAs (Figures 1A–C). Subsequently, size exclusion chromatography was used to remove aggregates or misfolded species with different hydrodynamic volumes and to put the RNA samples into the crystallization buffer. The target RNAs typically eluted in a single sharp peak (Figure 1D). We deliberately avoided a concentrating step via ethanol or isopropanol precipitation, which can re-introduce aggregation. Via the outlined protocol, we obtained around 3.0 mg of tRNA-sized RNAs in pure form from 2 ml transcription reactions.


Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse.

Ganichkin OM, Anedchenko EA, Wahl MC - PLoS ONE (2011)

Non-denaturing RNA purification.(A) Elution profile of in vitro transcribed mouse tRNASec from a MonoQ column. Peak 1 – unincorporated rNTPs, T7 RNA polymerase and other proteins; Peak 2 – abortive synthesis transcripts; Peak 3 – desired RNA sample; Peak 4 – aggregates or higher molecular weight nucleic acids. The gradient (buffer B from 30 to 100%) is shown as a dashed line. (B) Denaturing SDS PAGE analysis of peak fractions from Peaks 1–3. T7 RNA polymerase and molecular weight markers (M) were loaded as references. Protein bands were stained with Coomassie. (C) Denaturing urea PAGE analysis of peak fractions eluted from the MonoQ column. S – crude transcription extract. RNA bands were stained with methylene blue. (D) Elution profile of mouse tRNASec from a Superdex 75 10/300 GL column.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101227&req=5

pone-0020032-g001: Non-denaturing RNA purification.(A) Elution profile of in vitro transcribed mouse tRNASec from a MonoQ column. Peak 1 – unincorporated rNTPs, T7 RNA polymerase and other proteins; Peak 2 – abortive synthesis transcripts; Peak 3 – desired RNA sample; Peak 4 – aggregates or higher molecular weight nucleic acids. The gradient (buffer B from 30 to 100%) is shown as a dashed line. (B) Denaturing SDS PAGE analysis of peak fractions from Peaks 1–3. T7 RNA polymerase and molecular weight markers (M) were loaded as references. Protein bands were stained with Coomassie. (C) Denaturing urea PAGE analysis of peak fractions eluted from the MonoQ column. S – crude transcription extract. RNA bands were stained with methylene blue. (D) Elution profile of mouse tRNASec from a Superdex 75 10/300 GL column.
Mentions: We PCR-assembled DNA fragments encompassing a T7 RNA polymerase promoter and the RNA coding region and inserted the constructs into plasmids. The plasmids served as templates for amplification of the promoter and insert regions, using reverse primers with two 2′-O-methyl modified nucleotides at their 5′-ends to avoid 3′-end heterogeneity of the RNA products in the subsequent transcription reactions [28], [29]. The modified PCR products served as templates for in vitro transcription by T7 RNA polymerase. After removal of the DNA template by incubation with RNase-free DNase, the sample was directly loaded on a strong anion exchange column. None of the proteins present in the reaction mixture bound to the column under conditions, which afforded efficient binding and separation of RNAs (Figures 1A–C). Subsequently, size exclusion chromatography was used to remove aggregates or misfolded species with different hydrodynamic volumes and to put the RNA samples into the crystallization buffer. The target RNAs typically eluted in a single sharp peak (Figure 1D). We deliberately avoided a concentrating step via ethanol or isopropanol precipitation, which can re-introduce aggregation. Via the outlined protocol, we obtained around 3.0 mg of tRNA-sized RNAs in pure form from 2 ml transcription reactions.

Bottom Line: Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop.Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome.Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Strukturbiochemie, Freie Universität Berlin, Berlin, Germany.

ABSTRACT

Background: Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood.

Methodology/principal findings: We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec).

Conclusions/significance: We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins.

Show MeSH