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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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tRNASec constructs for crystallization screening.RNA 1 represents full-length tRNASec. Canonical tRNA numbering was used throughout. Additional nucleotides (labeled with lower case Latin characters) and gaps (missing numbers) compared to the canonical tRNA numbering are indicated only in the scheme of RNA 1. RNAs 2 and 3 were created by site-directed mutagenesis and contain a UUCG (red) or a kissing loop (green) in place of the wt variable loop, respectively. Using the initial, mutated constructs, further DNA templates were generated for in vitro transcription, which allowed synthesis of tRNASec species with deletion of the 3′-GCCA end (RNAs 4, 5 and 6) or with substitution of the 3′-GCCA with a self-complementary 3′-GCGC overhang (RNAs 7, 8 and 9).
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pone-0020032-g003: tRNASec constructs for crystallization screening.RNA 1 represents full-length tRNASec. Canonical tRNA numbering was used throughout. Additional nucleotides (labeled with lower case Latin characters) and gaps (missing numbers) compared to the canonical tRNA numbering are indicated only in the scheme of RNA 1. RNAs 2 and 3 were created by site-directed mutagenesis and contain a UUCG (red) or a kissing loop (green) in place of the wt variable loop, respectively. Using the initial, mutated constructs, further DNA templates were generated for in vitro transcription, which allowed synthesis of tRNASec species with deletion of the 3′-GCCA end (RNAs 4, 5 and 6) or with substitution of the 3′-GCCA with a self-complementary 3′-GCGC overhang (RNAs 7, 8 and 9).

Mentions: Purified mouse tRNASec crystallized readily under many different crystallization conditions but crystals exhibited low diffraction quality. In order to obtain better diffracting crystal forms, we generated mutant tRNASec molecules, which exhibited novel crystal packing potentials or contained elements that were expected to increase their conformational stabilities (Figure 3). We attempted substitution of the variable loop with a kissing loop [30] (to allow dimer formation) or with a UUCG tetraloop (to increase thermodynamic stability), introduction of a self-complementary 3′-overhang at the acceptor arm (to allow dimerization) and deletion of the 3′-GCCA overhang (to enhance stacking capacity at this end of the molecule). All constructs were predicted to retain the key structural features of the wild type (wt) molecule.


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

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

tRNASec constructs for crystallization screening.RNA 1 represents full-length tRNASec. Canonical tRNA numbering was used throughout. Additional nucleotides (labeled with lower case Latin characters) and gaps (missing numbers) compared to the canonical tRNA numbering are indicated only in the scheme of RNA 1. RNAs 2 and 3 were created by site-directed mutagenesis and contain a UUCG (red) or a kissing loop (green) in place of the wt variable loop, respectively. Using the initial, mutated constructs, further DNA templates were generated for in vitro transcription, which allowed synthesis of tRNASec species with deletion of the 3′-GCCA end (RNAs 4, 5 and 6) or with substitution of the 3′-GCCA with a self-complementary 3′-GCGC overhang (RNAs 7, 8 and 9).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101227&req=5

pone-0020032-g003: tRNASec constructs for crystallization screening.RNA 1 represents full-length tRNASec. Canonical tRNA numbering was used throughout. Additional nucleotides (labeled with lower case Latin characters) and gaps (missing numbers) compared to the canonical tRNA numbering are indicated only in the scheme of RNA 1. RNAs 2 and 3 were created by site-directed mutagenesis and contain a UUCG (red) or a kissing loop (green) in place of the wt variable loop, respectively. Using the initial, mutated constructs, further DNA templates were generated for in vitro transcription, which allowed synthesis of tRNASec species with deletion of the 3′-GCCA end (RNAs 4, 5 and 6) or with substitution of the 3′-GCCA with a self-complementary 3′-GCGC overhang (RNAs 7, 8 and 9).
Mentions: Purified mouse tRNASec crystallized readily under many different crystallization conditions but crystals exhibited low diffraction quality. In order to obtain better diffracting crystal forms, we generated mutant tRNASec molecules, which exhibited novel crystal packing potentials or contained elements that were expected to increase their conformational stabilities (Figure 3). We attempted substitution of the variable loop with a kissing loop [30] (to allow dimer formation) or with a UUCG tetraloop (to increase thermodynamic stability), introduction of a self-complementary 3′-overhang at the acceptor arm (to allow dimerization) and deletion of the 3′-GCCA overhang (to enhance stacking capacity at this end of the molecule). All constructs were predicted to retain the key structural features of the wild type (wt) molecule.

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