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The functional exchangeability of pk- and k-turns in RNA structure.

Daldrop P, Masquida B, Lilley DM - RNA Biol (2013)

Bottom Line: We find that we can replace the k-turn of the SAM-I riboswitch with the pk-turn, such that the resulting RNA retains its ability to bind SAM, although with lower affinity.Thus, although the pk-turn cannot intrinsically fold into the kinked structure, it can be induced to fold correctly in context.And the pk-turn and k-turns can substitute functionally for one another.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Nucleic Acid Structure Research Group; MSI/WTB Complex; The University of Dundee; Dundee, UK.

ABSTRACT
Ribonuclease P RNA requires a sharply kinked RNA helix to make a loop-receptor interaction that creates the binding site for the substrate. In some forms of the ribozyme, this is accomplished by a k-turn, while others have a different element called the pk-turn. The structure of the pk-turn in RNase P of Thermotoga maritima is globally very similar to a k-turn, but lacks all the standard features of that structure, including long-range hydrogen bonds between the two helical arms. We show here that in an isolated RNA duplex, the pk-turn fails to adopt a tightly kinked structure, but rather is a flexible element. This suggests that the tertiary contacts of RNase P assist its folding into the required kinked structure. We find that we can replace the k-turn of the SAM-I riboswitch with the pk-turn, such that the resulting RNA retains its ability to bind SAM, although with lower affinity. We also find that we can replace the pk-turn of T. maritima RNase P with a standard k-turn (in either orientation) with retention of ribozyme activity. Thus, although the pk-turn cannot intrinsically fold into the kinked structure, it can be induced to fold correctly in context. And the pk-turn and k-turns can substitute functionally for one another.

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Figure 2. Analysis of ion-induced structural change in duplex RNA-containing a pk-turn studied by fluorescence resonance energy transfer in the steady-state. The pk-turn was centrally located (boxed in the sequence shown) within a 22 bp duplex of RNA terminally 5′-labeled with fluorescein (F, donor) and Cy3 (Cy, acceptor) fluorophores. FRET efficiency was measured in 90 mM Tris.borate (pH 8.3) with addition of MgCl2 to the indicated concentrations. The data are plotted as FRET efficiency (EFRET) as a function of Mg2+ concentration. Data for the SAM-I riboswitch k-turn are plotted (broken line) as the best fit to a two-state ion-induced transition, taken from.13 Note that there is no increase in EFRET for the pk-turn-containing RNA at any Mg2+ concentration.
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Figure 2: Figure 2. Analysis of ion-induced structural change in duplex RNA-containing a pk-turn studied by fluorescence resonance energy transfer in the steady-state. The pk-turn was centrally located (boxed in the sequence shown) within a 22 bp duplex of RNA terminally 5′-labeled with fluorescein (F, donor) and Cy3 (Cy, acceptor) fluorophores. FRET efficiency was measured in 90 mM Tris.borate (pH 8.3) with addition of MgCl2 to the indicated concentrations. The data are plotted as FRET efficiency (EFRET) as a function of Mg2+ concentration. Data for the SAM-I riboswitch k-turn are plotted (broken line) as the best fit to a two-state ion-induced transition, taken from.13 Note that there is no increase in EFRET for the pk-turn-containing RNA at any Mg2+ concentration.

Mentions: For this purpose, the pk-turn sequence was incorporated into the center of a short RNA double helix labeled with fluorescein (fluorescent donor) and Cy3 (acceptor) at the two 5′-termini. The FRET efficiency was then measured as a function of Mg2+ concentration (Fig. 2). Folding into a kinked geometry shortens the inter-fluorophore distance, leading to an increase in FRET efficiency (EFRET). The SAM-I k-turn undergoes an increase in EFRET as the concentration of Mg2+ is increased typical of many k-turns, showing the stabilization of the kinked geometry of the k-turn (broken line in Fig. 2). In marked contrast, however, the pk-turn exhibits a low value of EFRET that does not increase with addition of Mg2+ (points in Fig. 2) We conclude that in isolation, the pk-turn fails to adopt a kinked conformation that is stabilized by Mg2+ ions and, thus, it seems unable intrinsically to adopt the tightly kinked conformation. But since it has been shown to have a structure that is globally similar to a k-turn when part of the complete ribozyme, it is more likely to be a point of flexibility that can be stabilized in the kinked form by other influences such as tertiary interactions.


The functional exchangeability of pk- and k-turns in RNA structure.

Daldrop P, Masquida B, Lilley DM - RNA Biol (2013)

Figure 2. Analysis of ion-induced structural change in duplex RNA-containing a pk-turn studied by fluorescence resonance energy transfer in the steady-state. The pk-turn was centrally located (boxed in the sequence shown) within a 22 bp duplex of RNA terminally 5′-labeled with fluorescein (F, donor) and Cy3 (Cy, acceptor) fluorophores. FRET efficiency was measured in 90 mM Tris.borate (pH 8.3) with addition of MgCl2 to the indicated concentrations. The data are plotted as FRET efficiency (EFRET) as a function of Mg2+ concentration. Data for the SAM-I riboswitch k-turn are plotted (broken line) as the best fit to a two-state ion-induced transition, taken from.13 Note that there is no increase in EFRET for the pk-turn-containing RNA at any Mg2+ concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672288&req=5

Figure 2: Figure 2. Analysis of ion-induced structural change in duplex RNA-containing a pk-turn studied by fluorescence resonance energy transfer in the steady-state. The pk-turn was centrally located (boxed in the sequence shown) within a 22 bp duplex of RNA terminally 5′-labeled with fluorescein (F, donor) and Cy3 (Cy, acceptor) fluorophores. FRET efficiency was measured in 90 mM Tris.borate (pH 8.3) with addition of MgCl2 to the indicated concentrations. The data are plotted as FRET efficiency (EFRET) as a function of Mg2+ concentration. Data for the SAM-I riboswitch k-turn are plotted (broken line) as the best fit to a two-state ion-induced transition, taken from.13 Note that there is no increase in EFRET for the pk-turn-containing RNA at any Mg2+ concentration.
Mentions: For this purpose, the pk-turn sequence was incorporated into the center of a short RNA double helix labeled with fluorescein (fluorescent donor) and Cy3 (acceptor) at the two 5′-termini. The FRET efficiency was then measured as a function of Mg2+ concentration (Fig. 2). Folding into a kinked geometry shortens the inter-fluorophore distance, leading to an increase in FRET efficiency (EFRET). The SAM-I k-turn undergoes an increase in EFRET as the concentration of Mg2+ is increased typical of many k-turns, showing the stabilization of the kinked geometry of the k-turn (broken line in Fig. 2). In marked contrast, however, the pk-turn exhibits a low value of EFRET that does not increase with addition of Mg2+ (points in Fig. 2) We conclude that in isolation, the pk-turn fails to adopt a kinked conformation that is stabilized by Mg2+ ions and, thus, it seems unable intrinsically to adopt the tightly kinked conformation. But since it has been shown to have a structure that is globally similar to a k-turn when part of the complete ribozyme, it is more likely to be a point of flexibility that can be stabilized in the kinked form by other influences such as tertiary interactions.

Bottom Line: We find that we can replace the k-turn of the SAM-I riboswitch with the pk-turn, such that the resulting RNA retains its ability to bind SAM, although with lower affinity.Thus, although the pk-turn cannot intrinsically fold into the kinked structure, it can be induced to fold correctly in context.And the pk-turn and k-turns can substitute functionally for one another.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Nucleic Acid Structure Research Group; MSI/WTB Complex; The University of Dundee; Dundee, UK.

ABSTRACT
Ribonuclease P RNA requires a sharply kinked RNA helix to make a loop-receptor interaction that creates the binding site for the substrate. In some forms of the ribozyme, this is accomplished by a k-turn, while others have a different element called the pk-turn. The structure of the pk-turn in RNase P of Thermotoga maritima is globally very similar to a k-turn, but lacks all the standard features of that structure, including long-range hydrogen bonds between the two helical arms. We show here that in an isolated RNA duplex, the pk-turn fails to adopt a tightly kinked structure, but rather is a flexible element. This suggests that the tertiary contacts of RNase P assist its folding into the required kinked structure. We find that we can replace the k-turn of the SAM-I riboswitch with the pk-turn, such that the resulting RNA retains its ability to bind SAM, although with lower affinity. We also find that we can replace the pk-turn of T. maritima RNase P with a standard k-turn (in either orientation) with retention of ribozyme activity. Thus, although the pk-turn cannot intrinsically fold into the kinked structure, it can be induced to fold correctly in context. And the pk-turn and k-turns can substitute functionally for one another.

Show MeSH
Related in: MedlinePlus