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In vitro selection of a 5'-purine ribonucleotide transferase ribozyme.

Kang TJ, Suga H - Nucleic Acids Res. (2007)

Bottom Line: This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS.Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates.This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan.

ABSTRACT
Here we report in vitro selection of a novel ribozyme that catalyzes the 5'-nucleotidyl transfer reaction forming the 2'-5' phosphodiester bond. This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme a trans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5'-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by the trans-acting system and elucidated the chemistry to be 3'-->5' mononucleotide extension forming the 2'-5' phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

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Proposed secondary structures of M4 ribozyme and ligases. (A) M4 from this study. (B) Bartel's class II ligase ribozyme (25). (C) Ellington's L1 ligase ribozyme (26). (D) and (E) Joyce's R3C and cytidine-free ligase ribozyme, respectively (27,28). (F) Siverman's 7S11 RNA ligase DNA enzyme (30). Bases likely involved in catalysis are shown. Other parts of ribozyme sequences are shown in solid line; DNA parts are shown in thicker solid line. The hydroxyl nucleophile of incoming purine (R) or oligonucleotide substrate (dashed lines) attacks the α-phosphate on the ribozyme.
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Figure 6: Proposed secondary structures of M4 ribozyme and ligases. (A) M4 from this study. (B) Bartel's class II ligase ribozyme (25). (C) Ellington's L1 ligase ribozyme (26). (D) and (E) Joyce's R3C and cytidine-free ligase ribozyme, respectively (27,28). (F) Siverman's 7S11 RNA ligase DNA enzyme (30). Bases likely involved in catalysis are shown. Other parts of ribozyme sequences are shown in solid line; DNA parts are shown in thicker solid line. The hydroxyl nucleophile of incoming purine (R) or oligonucleotide substrate (dashed lines) attacks the α-phosphate on the ribozyme.

Mentions: During the course of mutation studies, we found that indispensable motifs in M4 reside in the regions of the 5′-overhang, P2–P3 junction and L3 loop (Figures 3 and 6A). Since M4 catalyzes essentially the same chemistry as RNA ligase ribozymes except for the directionality of extension, it is of interest to compare the secondary structure of M4 with those of reported ligase ribozymes. Four representative ligases are selected for comparison; Bartel's class II ligase, Ellington's L1 ligase, Joyce's R3C and cytidine-free ligases (Figure 6B–E) (25–28), all of which form a 2′–5′ phosphodiester linkages like M4 ribozyme. These four ligases share the structural features of 5′-overhang and internal guide sequence for the incoming oligonucleotide. An obvious similarity found in all ribozymes including M4 is that the junction domain likely consisting of a part of catalytic core is dominated with A and G, occasionally containing U (note that G is dominated at the 5′-end because the consecutive Gs generally give higher transcription efficiency and therefore such RNA pools have been often used for selection). This suggests that such AG-rich motifs are suitable for composing the tertiary space where 5′-α-phosphate is positioned to the 2′-OH of the incoming nucleotide. On the other hand, a significant difference between M4 and these ribozymes is that M4 lacks the internal guide sequence for specific base pairing. Probably, the P2–P3 junction and L3 region would be located in close proximity, creating a 3D space for bringing the incoming purine nucleotides to the active site via base-stacking interactions.Figure 6.


In vitro selection of a 5'-purine ribonucleotide transferase ribozyme.

Kang TJ, Suga H - Nucleic Acids Res. (2007)

Proposed secondary structures of M4 ribozyme and ligases. (A) M4 from this study. (B) Bartel's class II ligase ribozyme (25). (C) Ellington's L1 ligase ribozyme (26). (D) and (E) Joyce's R3C and cytidine-free ligase ribozyme, respectively (27,28). (F) Siverman's 7S11 RNA ligase DNA enzyme (30). Bases likely involved in catalysis are shown. Other parts of ribozyme sequences are shown in solid line; DNA parts are shown in thicker solid line. The hydroxyl nucleophile of incoming purine (R) or oligonucleotide substrate (dashed lines) attacks the α-phosphate on the ribozyme.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Proposed secondary structures of M4 ribozyme and ligases. (A) M4 from this study. (B) Bartel's class II ligase ribozyme (25). (C) Ellington's L1 ligase ribozyme (26). (D) and (E) Joyce's R3C and cytidine-free ligase ribozyme, respectively (27,28). (F) Siverman's 7S11 RNA ligase DNA enzyme (30). Bases likely involved in catalysis are shown. Other parts of ribozyme sequences are shown in solid line; DNA parts are shown in thicker solid line. The hydroxyl nucleophile of incoming purine (R) or oligonucleotide substrate (dashed lines) attacks the α-phosphate on the ribozyme.
Mentions: During the course of mutation studies, we found that indispensable motifs in M4 reside in the regions of the 5′-overhang, P2–P3 junction and L3 loop (Figures 3 and 6A). Since M4 catalyzes essentially the same chemistry as RNA ligase ribozymes except for the directionality of extension, it is of interest to compare the secondary structure of M4 with those of reported ligase ribozymes. Four representative ligases are selected for comparison; Bartel's class II ligase, Ellington's L1 ligase, Joyce's R3C and cytidine-free ligases (Figure 6B–E) (25–28), all of which form a 2′–5′ phosphodiester linkages like M4 ribozyme. These four ligases share the structural features of 5′-overhang and internal guide sequence for the incoming oligonucleotide. An obvious similarity found in all ribozymes including M4 is that the junction domain likely consisting of a part of catalytic core is dominated with A and G, occasionally containing U (note that G is dominated at the 5′-end because the consecutive Gs generally give higher transcription efficiency and therefore such RNA pools have been often used for selection). This suggests that such AG-rich motifs are suitable for composing the tertiary space where 5′-α-phosphate is positioned to the 2′-OH of the incoming nucleotide. On the other hand, a significant difference between M4 and these ribozymes is that M4 lacks the internal guide sequence for specific base pairing. Probably, the P2–P3 junction and L3 region would be located in close proximity, creating a 3D space for bringing the incoming purine nucleotides to the active site via base-stacking interactions.Figure 6.

Bottom Line: This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS.Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates.This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan.

ABSTRACT
Here we report in vitro selection of a novel ribozyme that catalyzes the 5'-nucleotidyl transfer reaction forming the 2'-5' phosphodiester bond. This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme a trans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5'-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by the trans-acting system and elucidated the chemistry to be 3'-->5' mononucleotide extension forming the 2'-5' phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

Show MeSH