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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 3. Analysis of the distribution of fluorescein fluorescent lifetime distributions for pk-turn and k-turn-containing RNA as a function of the presence or absence of Mg2+ ions. The same fluorescein, Cy3 pk-turn-containing RNA and an equivalent species containing the H. marismortui Kt-7 were used in these experiments.
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Figure 3: Figure 3. Analysis of the distribution of fluorescein fluorescent lifetime distributions for pk-turn and k-turn-containing RNA as a function of the presence or absence of Mg2+ ions. The same fluorescein, Cy3 pk-turn-containing RNA and an equivalent species containing the H. marismortui Kt-7 were used in these experiments.

Mentions: We have used time-resolved fluorescence measurements of the same donor-acceptor-labeled pk-turn RNA to gain some insight into the distribution of conformations that are present as a function of solution conditions. We have compared the results with comparable measurements on the standard k-turn Kt-7 of H. marismortui. The decay of fluorescein fluorescence following a rapid excitation pulse was fitted to obtain distributions of excited state lifetimes (Fig. 3). Kt-7 in the absence of Mg2+ ions exhibits a distribution that comprises a single peak, with a mean lifetime of 3.3 ns. This corresponds to EFRET = 0.21, i.e., the extended, unfolded structure expected for the k-turn in these conditions. Upon addition of 2 mM Mg2+ to the sample, the distribution becomes bimodal, with a narrowed peak of 3.3 ns, and a second distribution with a mean lifetime of 0.95 ns. The latter corresponds to EFRET = 0.77, in good agreement with single-molecule analysis of the folded conformation of the k-turn.24 Evidently, under these conditions, the solution contains similar fractions of folded and unfolded k-turn molecules.


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

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

Figure 3. Analysis of the distribution of fluorescein fluorescent lifetime distributions for pk-turn and k-turn-containing RNA as a function of the presence or absence of Mg2+ ions. The same fluorescein, Cy3 pk-turn-containing RNA and an equivalent species containing the H. marismortui Kt-7 were used in these experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Figure 3. Analysis of the distribution of fluorescein fluorescent lifetime distributions for pk-turn and k-turn-containing RNA as a function of the presence or absence of Mg2+ ions. The same fluorescein, Cy3 pk-turn-containing RNA and an equivalent species containing the H. marismortui Kt-7 were used in these experiments.
Mentions: We have used time-resolved fluorescence measurements of the same donor-acceptor-labeled pk-turn RNA to gain some insight into the distribution of conformations that are present as a function of solution conditions. We have compared the results with comparable measurements on the standard k-turn Kt-7 of H. marismortui. The decay of fluorescein fluorescence following a rapid excitation pulse was fitted to obtain distributions of excited state lifetimes (Fig. 3). Kt-7 in the absence of Mg2+ ions exhibits a distribution that comprises a single peak, with a mean lifetime of 3.3 ns. This corresponds to EFRET = 0.21, i.e., the extended, unfolded structure expected for the k-turn in these conditions. Upon addition of 2 mM Mg2+ to the sample, the distribution becomes bimodal, with a narrowed peak of 3.3 ns, and a second distribution with a mean lifetime of 0.95 ns. The latter corresponds to EFRET = 0.77, in good agreement with single-molecule analysis of the folded conformation of the k-turn.24 Evidently, under these conditions, the solution contains similar fractions of folded and unfolded k-turn molecules.

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