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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 4. Replacement of the SAM-I riboswitch k-turn with the pk-turn. (A) A scheme shows the secondary structure and tertiary interaction of the SAM-I riboswitch, with the SAM-binding site indicated. The natural k-turn has been replaced by the pk turn in this structure. (B) Plots showing the results of microcalorimetric analysis of ligand binding to the SAM-I riboswitch containing the pk-turn. The upper panel shows the raw data for sequential injections of 2 µl volumes of a 5 µM solution of SAM into a 1.4 ml volume of 96 µM RNA solution in 50 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2. This represents the differential of the total heat evolved for each SAM concentration (i.e., ∆H°). The lower panel presents the integrated heat data fitted to a single-site binding model (Eq. 1).
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Figure 4: Figure 4. Replacement of the SAM-I riboswitch k-turn with the pk-turn. (A) A scheme shows the secondary structure and tertiary interaction of the SAM-I riboswitch, with the SAM-binding site indicated. The natural k-turn has been replaced by the pk turn in this structure. (B) Plots showing the results of microcalorimetric analysis of ligand binding to the SAM-I riboswitch containing the pk-turn. The upper panel shows the raw data for sequential injections of 2 µl volumes of a 5 µM solution of SAM into a 1.4 ml volume of 96 µM RNA solution in 50 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2. This represents the differential of the total heat evolved for each SAM concentration (i.e., ∆H°). The lower panel presents the integrated heat data fitted to a single-site binding model (Eq. 1).

Mentions: We substituted the k-turn for the pk-turn in the Thermoanaerobacter tengcongensis SAM-I riboswitch by site-directed mutagenesis of the template plasmid, and transcribed the modified riboswitch RNA. ITC study shows that the pk-substituted riboswitch binds SAM in an exothermic reaction (Fig. 4), with a lowered affinity of 300 µM (Table 1). Thus, the pk-turn can clearly substitute for the k-turn with retention of activity, suggesting that it can accommodate the kinked structure required for the folding of the riboswitch. However, the binding affinity is 1,000-fold lower than for the natural riboswitch.


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

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

Figure 4. Replacement of the SAM-I riboswitch k-turn with the pk-turn. (A) A scheme shows the secondary structure and tertiary interaction of the SAM-I riboswitch, with the SAM-binding site indicated. The natural k-turn has been replaced by the pk turn in this structure. (B) Plots showing the results of microcalorimetric analysis of ligand binding to the SAM-I riboswitch containing the pk-turn. The upper panel shows the raw data for sequential injections of 2 µl volumes of a 5 µM solution of SAM into a 1.4 ml volume of 96 µM RNA solution in 50 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2. This represents the differential of the total heat evolved for each SAM concentration (i.e., ∆H°). The lower panel presents the integrated heat data fitted to a single-site binding model (Eq. 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Figure 4. Replacement of the SAM-I riboswitch k-turn with the pk-turn. (A) A scheme shows the secondary structure and tertiary interaction of the SAM-I riboswitch, with the SAM-binding site indicated. The natural k-turn has been replaced by the pk turn in this structure. (B) Plots showing the results of microcalorimetric analysis of ligand binding to the SAM-I riboswitch containing the pk-turn. The upper panel shows the raw data for sequential injections of 2 µl volumes of a 5 µM solution of SAM into a 1.4 ml volume of 96 µM RNA solution in 50 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2. This represents the differential of the total heat evolved for each SAM concentration (i.e., ∆H°). The lower panel presents the integrated heat data fitted to a single-site binding model (Eq. 1).
Mentions: We substituted the k-turn for the pk-turn in the Thermoanaerobacter tengcongensis SAM-I riboswitch by site-directed mutagenesis of the template plasmid, and transcribed the modified riboswitch RNA. ITC study shows that the pk-substituted riboswitch binds SAM in an exothermic reaction (Fig. 4), with a lowered affinity of 300 µM (Table 1). Thus, the pk-turn can clearly substitute for the k-turn with retention of activity, suggesting that it can accommodate the kinked structure required for the folding of the riboswitch. However, the binding affinity is 1,000-fold lower than for the natural riboswitch.

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