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Attenuation of loop-receptor interactions with pseudoknot formation.

Afonin KA, Lin YP, Calkins ER, Jaeger L - Nucleic Acids Res. (2011)

Bottom Line: Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others.Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor.Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA.

ABSTRACT
RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more 'G/C-rich' than their 'A/U-rich' natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for 'A/U-rich' GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

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Co-transcriptional assemblies of tectoRNA attenuators 1, 4, 7 and 9 in presence (or absence) of cognate GAAA probe. (A) Schematic illustrating the possible molecular states adopted by the tectoRNA attenuator system during its transcription from DNA templates (in blue) by T7 RNA polymerase (in green) at 37°C in presence of 10 mM Mg2+. (B) Native PAGE analysis of different tectoRNA attenuator transcription mixtures at various times in presence (+) or absence (−) of GAAA probe: co-transcriptional assembly is monitored by RNA body-labeling with α[P32]ATP and native PAGE is performed at 10°C and 10 mM Mg(OAc)2 after quenching the transcription with DNase as described in the ‘Materials and Methods’ section. See also Supplementary Figure S5.
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gkr926-F6: Co-transcriptional assemblies of tectoRNA attenuators 1, 4, 7 and 9 in presence (or absence) of cognate GAAA probe. (A) Schematic illustrating the possible molecular states adopted by the tectoRNA attenuator system during its transcription from DNA templates (in blue) by T7 RNA polymerase (in green) at 37°C in presence of 10 mM Mg2+. (B) Native PAGE analysis of different tectoRNA attenuator transcription mixtures at various times in presence (+) or absence (−) of GAAA probe: co-transcriptional assembly is monitored by RNA body-labeling with α[P32]ATP and native PAGE is performed at 10°C and 10 mM Mg(OAc)2 after quenching the transcription with DNase as described in the ‘Materials and Methods’ section. See also Supplementary Figure S5.

Mentions: We have also investigated how tectoRNA attenuation could occur during in vitro RNA transcription in isothermal conditions (37°C) (see ‘Materials and Methods’ section). While all the experiments described above were performed in conditions usually favoring thermodynamic control versus kinetic control, co-transcriptional assembly experiments should be more representative of folding and assembly processes taking place within the cell (48,49). During the linear phase of RNA transcription, three different types of products are observed on native PAGE: the RNA probe, the tectoRNA attenuator and the complex resulting from the inter-molecular assembly between the probe and attenuator molecules (Figure 6A and Supplementary Figure S5). Because of its smaller size, the probe product is transcribed in larger quantity than the attenuator product, explaining why a portion of it always remains unassembled. In presence of GAAA probe, the totality of attenuator 1 (and 10) products assembles to the probe (Figure 6B). In contrast, only ~60% of the attenuator 4 product forms a stable complex with the probe, suggesting that the remaining 40% is blocked into the PK conformation state (Figure 6B). In perfect agreement with previous data, attenuator 7 (and 7″) demonstrates full attenuation of tectoRNA assembly, while attenuator 9, which differs from molecule 7 by only two point mutations within its intra-molecular PK, assembles with the probe to its full extent (Figure 6B and Supplementary Figure S5). Interestingly, attenuators 14, 16 and 17 assemble with their cognate probe to form complexes with faster gel mobility than those obtained with attenuator 1, 9 and 10 (Supplementary Figure S5). We have observed that tectoRNA complexes with higher Kd's (or lower affinities) typically migrate faster at lower RNA concentrations than those with lower Kd's (or higher affinities) (15,23). This behavior has been described as resulting from monomers and heterodimers being in dynamic equilibrium (15,23). Our observation corroborates the fact that attenuators 14, 16 and 17 bind less efficiently their cognate probe than their corresponding HD_forming modules. In these attenuators, formation of a transient intra-molecular PK likely displaces the inter-molecular assembly equilibrium toward the monomers.Figure 6.


Attenuation of loop-receptor interactions with pseudoknot formation.

Afonin KA, Lin YP, Calkins ER, Jaeger L - Nucleic Acids Res. (2011)

Co-transcriptional assemblies of tectoRNA attenuators 1, 4, 7 and 9 in presence (or absence) of cognate GAAA probe. (A) Schematic illustrating the possible molecular states adopted by the tectoRNA attenuator system during its transcription from DNA templates (in blue) by T7 RNA polymerase (in green) at 37°C in presence of 10 mM Mg2+. (B) Native PAGE analysis of different tectoRNA attenuator transcription mixtures at various times in presence (+) or absence (−) of GAAA probe: co-transcriptional assembly is monitored by RNA body-labeling with α[P32]ATP and native PAGE is performed at 10°C and 10 mM Mg(OAc)2 after quenching the transcription with DNase as described in the ‘Materials and Methods’ section. See also Supplementary Figure S5.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3300017&req=5

gkr926-F6: Co-transcriptional assemblies of tectoRNA attenuators 1, 4, 7 and 9 in presence (or absence) of cognate GAAA probe. (A) Schematic illustrating the possible molecular states adopted by the tectoRNA attenuator system during its transcription from DNA templates (in blue) by T7 RNA polymerase (in green) at 37°C in presence of 10 mM Mg2+. (B) Native PAGE analysis of different tectoRNA attenuator transcription mixtures at various times in presence (+) or absence (−) of GAAA probe: co-transcriptional assembly is monitored by RNA body-labeling with α[P32]ATP and native PAGE is performed at 10°C and 10 mM Mg(OAc)2 after quenching the transcription with DNase as described in the ‘Materials and Methods’ section. See also Supplementary Figure S5.
Mentions: We have also investigated how tectoRNA attenuation could occur during in vitro RNA transcription in isothermal conditions (37°C) (see ‘Materials and Methods’ section). While all the experiments described above were performed in conditions usually favoring thermodynamic control versus kinetic control, co-transcriptional assembly experiments should be more representative of folding and assembly processes taking place within the cell (48,49). During the linear phase of RNA transcription, three different types of products are observed on native PAGE: the RNA probe, the tectoRNA attenuator and the complex resulting from the inter-molecular assembly between the probe and attenuator molecules (Figure 6A and Supplementary Figure S5). Because of its smaller size, the probe product is transcribed in larger quantity than the attenuator product, explaining why a portion of it always remains unassembled. In presence of GAAA probe, the totality of attenuator 1 (and 10) products assembles to the probe (Figure 6B). In contrast, only ~60% of the attenuator 4 product forms a stable complex with the probe, suggesting that the remaining 40% is blocked into the PK conformation state (Figure 6B). In perfect agreement with previous data, attenuator 7 (and 7″) demonstrates full attenuation of tectoRNA assembly, while attenuator 9, which differs from molecule 7 by only two point mutations within its intra-molecular PK, assembles with the probe to its full extent (Figure 6B and Supplementary Figure S5). Interestingly, attenuators 14, 16 and 17 assemble with their cognate probe to form complexes with faster gel mobility than those obtained with attenuator 1, 9 and 10 (Supplementary Figure S5). We have observed that tectoRNA complexes with higher Kd's (or lower affinities) typically migrate faster at lower RNA concentrations than those with lower Kd's (or higher affinities) (15,23). This behavior has been described as resulting from monomers and heterodimers being in dynamic equilibrium (15,23). Our observation corroborates the fact that attenuators 14, 16 and 17 bind less efficiently their cognate probe than their corresponding HD_forming modules. In these attenuators, formation of a transient intra-molecular PK likely displaces the inter-molecular assembly equilibrium toward the monomers.Figure 6.

Bottom Line: Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others.Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor.Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA.

ABSTRACT
RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more 'G/C-rich' than their 'A/U-rich' natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for 'A/U-rich' GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

Show MeSH
Related in: MedlinePlus