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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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Related in: MedlinePlus

Lead(II)-induced cleavage patterns for tectoRNA attenuators 7 and 9 in their monomeric and heterodimeric states. (A) 2D diagrams of tectoRNA attenuators with reported differential Pb(II) cleavage patterns in the monomeric (M) and heterodimeric (D) states. Phosphate positions in monomer 7 (M7) that show enhanced or reduced Pb(II) cleavage with respect to monomer 9 (M9) are indicated by red or blue arrows on the 2D diagram of 7, respectively. Phosphate positions in heterodimer 9 (D9) that show enhanced or reduced Pb(II) cleavage with respect to heterodimer 7 (D7) are indicated by red or blue arrows on the 2D diagram of 9, respectively. The size of the arrows is roughly proportionate to the difference in cleavage for M7 versus M9 or D9 versus D7. A star indicates the radiolabeled RNA 3′-end. (B) Pb(II) cleavage patterns of 32P radiolabeled molecules 7 and 9 either alone or bound to their non-radioactive cognate GAAA probe [as shown in (A)]. M and D correspond to monomer and dimer lanes, respectively. Cleavage experiments (indicated by Pb2+) were carried out as described in the Materials and Methods section; OH− indicates alkaline hydrolysis ladder; T1 indicates RNase T1 digestion. (C) Superposed lead cleavage profiles for monomers 7 and 9 (top) and for the corresponding heterodimers in presence of GAAA probe (bottom). Black dots indicate positions used for normalization. Similar results are obtained by comparing attenuators 7 and 8 (Supplementary Figure S3).
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gkr926-F4: Lead(II)-induced cleavage patterns for tectoRNA attenuators 7 and 9 in their monomeric and heterodimeric states. (A) 2D diagrams of tectoRNA attenuators with reported differential Pb(II) cleavage patterns in the monomeric (M) and heterodimeric (D) states. Phosphate positions in monomer 7 (M7) that show enhanced or reduced Pb(II) cleavage with respect to monomer 9 (M9) are indicated by red or blue arrows on the 2D diagram of 7, respectively. Phosphate positions in heterodimer 9 (D9) that show enhanced or reduced Pb(II) cleavage with respect to heterodimer 7 (D7) are indicated by red or blue arrows on the 2D diagram of 9, respectively. The size of the arrows is roughly proportionate to the difference in cleavage for M7 versus M9 or D9 versus D7. A star indicates the radiolabeled RNA 3′-end. (B) Pb(II) cleavage patterns of 32P radiolabeled molecules 7 and 9 either alone or bound to their non-radioactive cognate GAAA probe [as shown in (A)]. M and D correspond to monomer and dimer lanes, respectively. Cleavage experiments (indicated by Pb2+) were carried out as described in the Materials and Methods section; OH− indicates alkaline hydrolysis ladder; T1 indicates RNase T1 digestion. (C) Superposed lead cleavage profiles for monomers 7 and 9 (top) and for the corresponding heterodimers in presence of GAAA probe (bottom). Black dots indicate positions used for normalization. Similar results are obtained by comparing attenuators 7 and 8 (Supplementary Figure S3).

Mentions: Additional structural evidences for intra-molecular PK formation in attenuator 7 are provided by lead cleavage experiments (Figure 4 and Supplementary Figure S3). Lead is widely used as a conformational probe for RNA because it preferentially cleaves the phosphodiester backbone in flexible regions or non-canonically paired motifs of RNA molecules (15,22,23,25,46). Irrespective from absence or presence of the GAAA probe, the 3′ strand of the 11nt_GU receptor of attenuator 7 is strongly protected toward cleavage in comparison to the one of molecules 8 (or 9). Additionally, the 5′ PK strand within the PK-forming module of 7 also shows enhanced protection toward lead cleavage relative to 8 (or 9). This strongly suggests that the PK is formed in attenuator 7 but not in 8 (or 9). In contrast, in presence of the GAAA probe, molecules 8 and 9 display partial protection of the receptor and tetraloop regions from the HD-forming module, corroborating their assembly with the probe.Figure 4.


Attenuation of loop-receptor interactions with pseudoknot formation.

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

Lead(II)-induced cleavage patterns for tectoRNA attenuators 7 and 9 in their monomeric and heterodimeric states. (A) 2D diagrams of tectoRNA attenuators with reported differential Pb(II) cleavage patterns in the monomeric (M) and heterodimeric (D) states. Phosphate positions in monomer 7 (M7) that show enhanced or reduced Pb(II) cleavage with respect to monomer 9 (M9) are indicated by red or blue arrows on the 2D diagram of 7, respectively. Phosphate positions in heterodimer 9 (D9) that show enhanced or reduced Pb(II) cleavage with respect to heterodimer 7 (D7) are indicated by red or blue arrows on the 2D diagram of 9, respectively. The size of the arrows is roughly proportionate to the difference in cleavage for M7 versus M9 or D9 versus D7. A star indicates the radiolabeled RNA 3′-end. (B) Pb(II) cleavage patterns of 32P radiolabeled molecules 7 and 9 either alone or bound to their non-radioactive cognate GAAA probe [as shown in (A)]. M and D correspond to monomer and dimer lanes, respectively. Cleavage experiments (indicated by Pb2+) were carried out as described in the Materials and Methods section; OH− indicates alkaline hydrolysis ladder; T1 indicates RNase T1 digestion. (C) Superposed lead cleavage profiles for monomers 7 and 9 (top) and for the corresponding heterodimers in presence of GAAA probe (bottom). Black dots indicate positions used for normalization. Similar results are obtained by comparing attenuators 7 and 8 (Supplementary Figure S3).
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Related In: Results  -  Collection

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gkr926-F4: Lead(II)-induced cleavage patterns for tectoRNA attenuators 7 and 9 in their monomeric and heterodimeric states. (A) 2D diagrams of tectoRNA attenuators with reported differential Pb(II) cleavage patterns in the monomeric (M) and heterodimeric (D) states. Phosphate positions in monomer 7 (M7) that show enhanced or reduced Pb(II) cleavage with respect to monomer 9 (M9) are indicated by red or blue arrows on the 2D diagram of 7, respectively. Phosphate positions in heterodimer 9 (D9) that show enhanced or reduced Pb(II) cleavage with respect to heterodimer 7 (D7) are indicated by red or blue arrows on the 2D diagram of 9, respectively. The size of the arrows is roughly proportionate to the difference in cleavage for M7 versus M9 or D9 versus D7. A star indicates the radiolabeled RNA 3′-end. (B) Pb(II) cleavage patterns of 32P radiolabeled molecules 7 and 9 either alone or bound to their non-radioactive cognate GAAA probe [as shown in (A)]. M and D correspond to monomer and dimer lanes, respectively. Cleavage experiments (indicated by Pb2+) were carried out as described in the Materials and Methods section; OH− indicates alkaline hydrolysis ladder; T1 indicates RNase T1 digestion. (C) Superposed lead cleavage profiles for monomers 7 and 9 (top) and for the corresponding heterodimers in presence of GAAA probe (bottom). Black dots indicate positions used for normalization. Similar results are obtained by comparing attenuators 7 and 8 (Supplementary Figure S3).
Mentions: Additional structural evidences for intra-molecular PK formation in attenuator 7 are provided by lead cleavage experiments (Figure 4 and Supplementary Figure S3). Lead is widely used as a conformational probe for RNA because it preferentially cleaves the phosphodiester backbone in flexible regions or non-canonically paired motifs of RNA molecules (15,22,23,25,46). Irrespective from absence or presence of the GAAA probe, the 3′ strand of the 11nt_GU receptor of attenuator 7 is strongly protected toward cleavage in comparison to the one of molecules 8 (or 9). Additionally, the 5′ PK strand within the PK-forming module of 7 also shows enhanced protection toward lead cleavage relative to 8 (or 9). This strongly suggests that the PK is formed in attenuator 7 but not in 8 (or 9). In contrast, in presence of the GAAA probe, molecules 8 and 9 display partial protection of the receptor and tetraloop regions from the HD-forming module, corroborating their assembly with the probe.Figure 4.

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