This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.
Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.
Prerequisite before initiating the experiment:
1. tRNAAsp Preparation
This procedure describes an in vitro run-off transcription which can be used for synthesis of any tRNA. This procedure is an adaptation of the ribozyme method published previously.9 For this purpose a template was designed consisting of the T7 promoter and the complementary sequences of a self-cleaving hammerhead ribozyme and the required tRNA. Transcription of this template results in a product cleaving itself at the 5`-end in a way that the full-length tRNA is obtained with a 5`-OH instead of a phosphorylated 5`-end. The full-length tRNA can be purified from cleaved hammerhead ribozyme and uncleaved products by urea-PAGE.
Troubleshooting:
2. Expression and Purification of the Recombinant E. histolytica Dnmt2 (Ehmeth) Protein in E. coli BL21
This protocol is also suitable for the preparation of Dnmt2 proteins from human 10 and Drosophila.11
Notice : to insure the preparation of an active enzyme, all purification steps should be done on ice and DTT should be freshly prepared and added to the storage buffer. This insures an active enzyme for 6 months.
3. In vitro TRNA Methylation Assay
Solutions and reagents that should be prepared:
The assay:
4. Troubleshooting
5. Representative Results
Reagent | Final Concentration | PCR – program | |||||
Gentherm PCR Buffer | 1X | 1. | 90°C | 2 min | |||
MgCl2 | 3.0 mM | —————————— | —————————— | ||||
dNTP mix | 1.2 mM | 2. | 90°C | 30 s | 5. | 90°C | 30 s |
Template DNA | 15.0 nM | 3. | 54°C | 30 s | 6. | 60°C | 30 s |
Forward Primer | 3.0 µM | 4. | 72°C | 45 s | 7. | 72°C | 45 s |
Reverse Primer | 3.0 µM | 2.-4. 10 cycles | 5.-7. 25 cycles | ||||
Taq Polymerase | 0.1 U/µl | —————————— | —————————— | ||||
H2O | Ad 50 µl | lid 95°C | 8. | 72°C | 3 min |
Table 1: Pipette scheme for PCR and PCR program.
A Negative control (Ehmeth only) |
B Negative control (tRNA only) |
C Positive control with human Dnmt2 |
D Standard Reaction (Enz+Subst+Cof) |
E Total value of the AdoMet on the filter |
|
tRNA | – | (3 μg) | (3 μg) | (3 μg) | – |
hDnmt2 | – | (2.48 μg) | – | ||
Ehmeth | (2.48 μg) | (2.48 μg) | |||
MB | 8 | 8 | 8 | 8 | – |
DTT | 4 | 4 | 4 | 4 | – |
AdoMet | 4 | 4 | 4 | 4 | 1 |
ddw | – | ||||
Total | 40 μL | 40 μL | 40 μL | 40 μL | 1 μL |
Table 2: Example of pipeting scheme. Each column in the scheme represents one sample.
* Both Ehmeth and hDnmt2 were fused to a GST tag with an estimated protein size of 62 kDa.
A- Negative control that includes Ehmeth only.
B- Negative control that includes tRNA only.
C- Positive control with human Dnmt2. This enzyme has ten time more activity than Ehmeth
D- Standard reaction that includes the enzyme, the substrate and the cofactor
E- Total value of the AdoMet on the filter. This value is used for the calculation of the percentage of methyl group incorporated.
A Negative control (Ehmeth only) |
B Negative control (tRNA only) |
C Positive control with human Dnmt2 |
D Standard Reaction (Enz+Subst+Cof) |
E Total value of the AdoMet on the filter |
|
cpm | 200 | 300 | 6700 | 1284 | 51653 |
Table 3: Example of values read by the scintillation counter. Each column in the scheme represents one sample from Table 1.
Figure 1: (A) T7 transcription template and primers for in vitro transcription. All sequences are written in 5′ to 3′ orientation. Colorcode: see text. (B) Scheme of product formation during transcription. The RNA transcript is synthesized by T7 RNA polymerase. Hammerhead ribozyme and tRNA fold into their respective 2D-structures and the ribozyme cleaves itself of the tRNA leaving a hydroxyl group at their 5′-end (adapted from 9).
Figure 2: PCR reaction control on a 1.3% agarose gel prestained with 1X GelRed. PCR reactions on two different templates are shown alongside a 100 basepairs plus ladder. Controls include a “template only” lane and reactions in which the reverse primer was omitted.
Figure 3: UV-shadowing. A PAGE separation of two transcription mixtures is shown. Bands of precursor transcripts (before cleavage), full length tRNA and hammerhead ribozyme become visible as shadows since they prevent UV light from reaching the fluorescent TLC plate. Total tRNA from E. coli is used as size marker.
In this presentation, we illustrated how to prepare active components for the measure of the tRNA methyltransferase activity of E. histolytica Ehmeth enzyme. This procedure can serve as a starting point and be easily adapted for the measure of other tRNA methyltransferases. It is important to follow the procedures that preserve the quality and the integrity of the tRNA substrate and of the tRNA methyltransferase protein to insure an optimal measure of the enzymatic activity.
The authors have nothing to disclose.
This study was supported by grants from the Israel Science Foundation and the Rappaport Family Institute for Research in the Medical Sciences, and the Deutsche Forschungsgemeinschaft (DFG).
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Taq Polymerase (5 U/μl) | Rapidozym, Berlin | GEN-003-1000 | ||
10X Gentherm PCR Buffer | Rapidozym, Berlin | With GEN-003-1000 | ||
MgCl2 (50 mM) | Rapidozym, Berlin | With GEN-003-1000 | ||
dNTP mix (10 mM) | Fermentas, St. Leon-Rot | R0191 | ||
Oligo nucleotides | IBA, Göttingen | Customized | ||
NTP mix (25mM) | Fermentas, St. Leon-Rot | R0481 | ||
GelRed Nucleic Acid Stain 3X in water | Biotium | 41001 | ||
Dithiotreitol | Fermentas, St. Leon-Rot | R0861 | ||
Spermidine | Sigma, Germany | S0266 | ||
Pall Nanosep 0.45 μm | Sigma, Germany | Z722014 | ||
Dichlorodimethylsilane | Sigma, Germany | 40140 | ||
Ethanol (Ph. Eur.) | Roth, Karlsruhe | 5054.3 | ||
Tris HCl | Roth, Karlsruhe | 9090.3 | ||
Tris | Roth, Karlsruhe | AE15.1 | ||
BSA | Fermentas, St. Leon-Rot | B14 | ||
Triton X-100 | Sigma, Germany | T8787 | ||
TEMED | Roth, Karlsruhe | 2367.1 | ||
Ammonium Peroxodisulfate | Roth, Karlsruhe | 9592.2 | ||
Rotiphorese Sequencing gel concentrate | Roth, Karlsruhe | 3043.1 | ||
Rotiphorese Sequencing gel diluent | Roth, Karlsruhe | 3047.1 | ||
Rotiphorese Sequencing gel buffer concentrate | Roth, Karlsruhe | 3050.1 | ||
Rotiphorese 10X TBE-buffer | Roth, Karlsruhe | 3061.2 | ||
DEPC | Sigma | D5758 | ||
RNAse ExTERMINATOR | Biological Industry Beit Haemek | 01-895-1B | ||
Ampicillin | Sigma | A0166 | ||
IPTG | Sigma | I6758 | ||
PMSF | Sigma | P7626 | ||
Lysozyme | Sigma | L7651 | ||
Leupeptine | Sigma | L2884 | ||
Benzonase Nuclease | Novagen | 70746-3 | ||
BugBuster protein extraction reagent | Novagen | 70921-3 | ||
Glutathione-agarose resin | Sigma | G4510 | ||
L-glutathione reduced | Sigma | G4251 | ||
AdoMet | New England Biolabs | B9003 | ||
AdoMet 3H | PerkinElmer | NET155250UC | ||
Scintillation Cocktail | CytoScint | 882453 |