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Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U.

Pearson D, Carell T - Nucleic Acids Res. (2011)

Bottom Line: Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process.These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations.This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

ABSTRACT
Transfer RNA (tRNA) contains a number of complex 'hypermodified' nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient 'assembly line' that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm(5)s(2)U nucleoside in Escherichia coli tRNA(Glu), which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a K(m) value of 600 nM and k(cat) 0.34 s(-1) for the first step, and K(m) 70 nM and k(cat) 0.31 s(-1) for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.

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Overexpressed tRNAGlu from a ΔMnmC E. coli strain. (a) HPLC of total extracted tRNA. HPLC buffers: 100 mM Tris pH 8, 50 → 150 mM MgCl2. The four major modivariants are labelled 1–4. Each was isolated by HPLC for subsequent MS analysis of modified nucleosides. (b) Representative purified tRNA (tRNA 5, see below) after anion-exchange purifications at pH 5 and 8. HPLC buffers: 100 mM Tris pH 8, 50 mM MgCl2, 0 → 500 mM NaCl. [Note: The MgCl2 and NaCl gradients in (a) and (b) were used interchangeably for analytical purposes] (c) Diagrams showing the modified nucleosides present in tRNAs 1–5 and those present in tRNAs 5 and 6, the products of reaction of tRNA 1 with MnmC (see below). Nucleosides labelled in red (or orange or yellow) were identified by MS (Figure 2 and Supplementary Figure S1). The presence of  nucleosides labelled in grey was not confirmed. tRNA 1 contains cmnm5s2U34 and all other expected modifications, while tRNA 2–4 are less fully modified at anticodon–stem loop postions as shown. The nm5s2U and mnm5s2U modifications obtained by reaction with MnmC are shown in orange and yellow, respectively, to differentiate from cmnm5s2U.
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Figure 1: Overexpressed tRNAGlu from a ΔMnmC E. coli strain. (a) HPLC of total extracted tRNA. HPLC buffers: 100 mM Tris pH 8, 50 → 150 mM MgCl2. The four major modivariants are labelled 1–4. Each was isolated by HPLC for subsequent MS analysis of modified nucleosides. (b) Representative purified tRNA (tRNA 5, see below) after anion-exchange purifications at pH 5 and 8. HPLC buffers: 100 mM Tris pH 8, 50 mM MgCl2, 0 → 500 mM NaCl. [Note: The MgCl2 and NaCl gradients in (a) and (b) were used interchangeably for analytical purposes] (c) Diagrams showing the modified nucleosides present in tRNAs 1–5 and those present in tRNAs 5 and 6, the products of reaction of tRNA 1 with MnmC (see below). Nucleosides labelled in red (or orange or yellow) were identified by MS (Figure 2 and Supplementary Figure S1). The presence of nucleosides labelled in grey was not confirmed. tRNA 1 contains cmnm5s2U34 and all other expected modifications, while tRNA 2–4 are less fully modified at anticodon–stem loop postions as shown. The nm5s2U and mnm5s2U modifications obtained by reaction with MnmC are shown in orange and yellow, respectively, to differentiate from cmnm5s2U.

Mentions: In order to prepare selectively under-modified tRNAGlu, we assembled a tRNA overexpression system (25) using an E. coli strain that lacks the MnmC gene. As the expression construct, the E. coli tRNAGlu gene was cloned into a protein expression plasmid (pSGAT2) containing a T7 promoter for inducible expression. An additional T7 promoter was added directly 5′ to the tRNA gene in order to expedite tRNA processing (as the RNA transcripts from this promoter should begin at the first position of the tRNA and therefore not require enzymatic 5′-cleavage). To obtain an expression strain lacking MnmC, we used the recombineering method (23) to replace the MnmC gene in T7 expression cells with an antibiotic resistance cassette. The tRNA expression plasmid was then used to transform ΔMnmC cells, from which tRNAGlu was expressed and isolated by phenol extraction (6). The major tRNA ‘modivariants’ (10) obtained were then separated by anion-exchange HPLC (Figure 1a and b) then partially sequenced by MALDI-MS analysis of RNase digests (Figure 2). All major peaks in the HPLC chromatogram were found to be tRNAGlu based on this MALDI sequencing (Figure 2a–c and Supplementary Figure S1), showing that a very high level of expression was sustained by the cells. A single modivariant (tRNA 1, Figure 1a and c) was found to contain the desired cmnm5s2U modification (Figure 2c) as well as the other expected mass-detectable tRNAGlu modifications (T and m2A, Figure 2a–c). The presence of the two expected pseudouridine () nucleotides in this tRNA was then confirmed by reaction with acrylonitrile followed by RNase/MS analysis (Supplementary Figure S2) (24). The other modivariants obtained lacked anticodon–stem loop modifications (Supplementary Figure S1), as labelled in Figure 1c.Figure 1.


Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U.

Pearson D, Carell T - Nucleic Acids Res. (2011)

Overexpressed tRNAGlu from a ΔMnmC E. coli strain. (a) HPLC of total extracted tRNA. HPLC buffers: 100 mM Tris pH 8, 50 → 150 mM MgCl2. The four major modivariants are labelled 1–4. Each was isolated by HPLC for subsequent MS analysis of modified nucleosides. (b) Representative purified tRNA (tRNA 5, see below) after anion-exchange purifications at pH 5 and 8. HPLC buffers: 100 mM Tris pH 8, 50 mM MgCl2, 0 → 500 mM NaCl. [Note: The MgCl2 and NaCl gradients in (a) and (b) were used interchangeably for analytical purposes] (c) Diagrams showing the modified nucleosides present in tRNAs 1–5 and those present in tRNAs 5 and 6, the products of reaction of tRNA 1 with MnmC (see below). Nucleosides labelled in red (or orange or yellow) were identified by MS (Figure 2 and Supplementary Figure S1). The presence of  nucleosides labelled in grey was not confirmed. tRNA 1 contains cmnm5s2U34 and all other expected modifications, while tRNA 2–4 are less fully modified at anticodon–stem loop postions as shown. The nm5s2U and mnm5s2U modifications obtained by reaction with MnmC are shown in orange and yellow, respectively, to differentiate from cmnm5s2U.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Overexpressed tRNAGlu from a ΔMnmC E. coli strain. (a) HPLC of total extracted tRNA. HPLC buffers: 100 mM Tris pH 8, 50 → 150 mM MgCl2. The four major modivariants are labelled 1–4. Each was isolated by HPLC for subsequent MS analysis of modified nucleosides. (b) Representative purified tRNA (tRNA 5, see below) after anion-exchange purifications at pH 5 and 8. HPLC buffers: 100 mM Tris pH 8, 50 mM MgCl2, 0 → 500 mM NaCl. [Note: The MgCl2 and NaCl gradients in (a) and (b) were used interchangeably for analytical purposes] (c) Diagrams showing the modified nucleosides present in tRNAs 1–5 and those present in tRNAs 5 and 6, the products of reaction of tRNA 1 with MnmC (see below). Nucleosides labelled in red (or orange or yellow) were identified by MS (Figure 2 and Supplementary Figure S1). The presence of nucleosides labelled in grey was not confirmed. tRNA 1 contains cmnm5s2U34 and all other expected modifications, while tRNA 2–4 are less fully modified at anticodon–stem loop postions as shown. The nm5s2U and mnm5s2U modifications obtained by reaction with MnmC are shown in orange and yellow, respectively, to differentiate from cmnm5s2U.
Mentions: In order to prepare selectively under-modified tRNAGlu, we assembled a tRNA overexpression system (25) using an E. coli strain that lacks the MnmC gene. As the expression construct, the E. coli tRNAGlu gene was cloned into a protein expression plasmid (pSGAT2) containing a T7 promoter for inducible expression. An additional T7 promoter was added directly 5′ to the tRNA gene in order to expedite tRNA processing (as the RNA transcripts from this promoter should begin at the first position of the tRNA and therefore not require enzymatic 5′-cleavage). To obtain an expression strain lacking MnmC, we used the recombineering method (23) to replace the MnmC gene in T7 expression cells with an antibiotic resistance cassette. The tRNA expression plasmid was then used to transform ΔMnmC cells, from which tRNAGlu was expressed and isolated by phenol extraction (6). The major tRNA ‘modivariants’ (10) obtained were then separated by anion-exchange HPLC (Figure 1a and b) then partially sequenced by MALDI-MS analysis of RNase digests (Figure 2). All major peaks in the HPLC chromatogram were found to be tRNAGlu based on this MALDI sequencing (Figure 2a–c and Supplementary Figure S1), showing that a very high level of expression was sustained by the cells. A single modivariant (tRNA 1, Figure 1a and c) was found to contain the desired cmnm5s2U modification (Figure 2c) as well as the other expected mass-detectable tRNAGlu modifications (T and m2A, Figure 2a–c). The presence of the two expected pseudouridine () nucleotides in this tRNA was then confirmed by reaction with acrylonitrile followed by RNase/MS analysis (Supplementary Figure S2) (24). The other modivariants obtained lacked anticodon–stem loop modifications (Supplementary Figure S1), as labelled in Figure 1c.Figure 1.

Bottom Line: Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process.These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations.This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

ABSTRACT
Transfer RNA (tRNA) contains a number of complex 'hypermodified' nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient 'assembly line' that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm(5)s(2)U nucleoside in Escherichia coli tRNA(Glu), which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a K(m) value of 600 nM and k(cat) 0.34 s(-1) for the first step, and K(m) 70 nM and k(cat) 0.31 s(-1) for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.

Show MeSH
Related in: MedlinePlus