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The MagneHis™ Protein Purification System (Cat.# V8550) was used to construct an artificial elongation complex containing a His-tagged T7 RNA polymerase. The elongation complex was used to study transcription elongation of platinum crosslinked DNA templates with the polymerase still bound to the MagneHis™ Ni-Particles. |
Introduction
The MagneHis™ Protein Purification System provides a simple, rapid and reliable method for
purifying polyhistidine-tagged proteins. The affinity of histidine residues for immobilized nickel allows for the selective purification of proteins that contain histidine tags
(1,2). His-tagged proteins bind to paramagnetic precharged nickel particles (MagneHis™ Ni-Particles) and
are captured using a magnetic stand. The bound protein can be washed and eluted.
In this study, MagneHis™ Ni-Particles were used to synthesize and purify a
transcription elongation complex containing a His-tagged RNA polymerase.
Platinum anticancer drugs, such as cisplatin, are used to successfully treat various types of cancers. Although the complete mechanism by which cisplatin
selectively kills cancer cells is not completely understood,
cisplatin-DNA adducts inhibit transcription by blocking RNA polymerase. In Jung's and Lippard's study, they isolated and studied T7 RNA polymerase complexes stalled by specific cisplatin-DNA adducts in vitro.
Since there can be multiple transcription initiation sites verifying the exact locations where transcription stalls based on transcript
size is difficult. This study used promoter-independent transcription to identify exact stop
sites caused by platinum-DNA adducts under different conditions.
Assembly of Artificial Elongation Complex
The artificial elongation complex consists of T7 RNA Polymerase associated wth a DNA:RNA hybrid carrying a platinum lesion. The complex was assembled in several steps using MagneHis™ Particles to isolate the complex between steps.
Step 1-Creation of a DNA:RNA hybrid associated with T7 RNA Polymerase. A 5´-phosphorylated PI T50 deoxyoligonucleotide was annealed with RNA8 (Dharmacon), labeled with 32P at the 5´-end, by heating at 50°C and cooling slowly to room temperature (Figure 1A). An 8pmol portion of this annealed probe was mixed with an equimolar quantity of His-tagged T7 RNA polymerase in a total volume of 20µl of transcription buffer (40mM Tris-HCl [pH 7.9], 6mM MgCl2, 2mM spermidine and 10mM NaCl). Following a 15-minute incubation at 30°C, 200pmol of PI C59 was added to the binding solution, and incubation was continued for an additional 10 minutes (Figure 1A).
Step 2-Formation of stable complex. This artificial elongation complex containing the 8-nucleotide (nt) RNA segment (EC RNA8) was extended by 6nt to form a more stable complex containing a 14nt RNA segment (EC RNA14) by incubation with 10µM ATP and GTP. The elongation complex was isolated from excess probe and NTPs by immobilization onto MagneHis™ Ni-Particles and was washed three times with transcription buffer (Figure 1B). Approximately 3pmol of elongation complex was obtained.
Step 3-Insertion of platinum adduct. An 86bp double-stranded DNA fragment with a 3´-overhang carrying a platinum intrastrand cross-link was prepared by annealing and ligating four oligonucleotides (Figure 1C).
Step 4-Ligation of "elongation complex" fragment to the "platinum adduct" fragment. Two picomoles of the fragment containing a specific platinum crosslink (not present in the control) was added to the MagneHis™-immobilized RNA14 (1pmol) elongation complex in 10µl of transcription buffer containing T4 DNA ligase (2.5 units), 0.5mM ATP, and 1% polyethylene glycol 8000. The ligation reaction was carried out at 12°C for 1 hour (Figure 1D). The T-95 strand of the 86bp probe was labeled at the 5´-end with 32P to test for proper ligation (note the 145bp band in Figure 2A).
Step 5- In vitro transcription. The resulting EC RNA14 complexes on the 145bp DNA probe (PI 145) were washed extensively with transcription buffer. Transcription buffer containing the indicated concentrations of NTPs was added to initiate transcription elongation (Figure 1E).
Promoter-Independent In Vitro Transcription
Figure 2 shows the results of transcription assays using the assembled elongation complex with two different platinum adducts under varying temperatures and NTP concentrations. The 133 nucleotide run-off transcript is present in the "no platinum" control, but no 133nt band is visible with either platinum adduct, indicating stalling of most of the elongation complexes (Figure 2A). Two different cisplatin adducts crosslinking different nucleotides on the template strand were used: a 1,2-intrastrand d(GpG) crosslink and a 1,3-intrastrand d(GpTpG) crosslink (Figure 2C). At low concentrations of NTPs, the polymerase stops before the d(GpG) crosslink, generating a 62nt transcript (Figure 2B). Higher NTP concentrations drive the polymerase into the crosslinked region. Even at low NTP concentrations, the polymerase is capable of reaching the first G in the d(GpTpG) adduct. At high NTP concentrations, the polymerase reaches the T residue in the crosslinked region.
Conclusions
His-tagged proteins are commonly used as a tool to isolate proteins from E.
coli for analysis. This study demonstrates the novel use of
His-tagging and MagneHis™ Ni-Particles to create, isolate, and study a
biomolecular complex in vitro. The authors used the MagneHis™ technology for easy
purification between steps in the synthesis of a promoter-independent elongation complex. The
complex was used to study transcription at the binding site of different platinum
adducts in response to various conditions. Only a portion of Jung's and Lippard's
findings are reviewed in this eNotes article. Please see the original article
for full details of their study.
Reference
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Figure 1. Steps in the assembly of an artificial elongation factor for promoter-independent transcription. "Bead" represents MagneHis™ Ni-Particle. Reprinted with the kind permission of Dr. Stephen J. Lippard, MIT, and the American Society for Biochemistry and Molecular Biology from Lippard and Jung (2003) J. Biol. Chem. 278, 52084. |
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Figure 2. Gel electrophoresis analysis of promoter-independent transcription on templates containing a cisplatin 1,2-d(GpG) or 1,3-d(GpTpG) crosslink. Transcription from EC RNA14 on undamaged, immobilized templates (No Pt) proceeded to EC RNA48 following the addition of 50µM ATP, CTP, and GTP. EC RNA62 was produced by adding 50µM ATP, GTP, and UTP to the EC RNA48 product after washing. A 50µM portion of NTP was used to generate 133 residues of run-off RNA transcripts from EC RNA14 using the No Pt template. The EC RNA14 complex formed on templates containing a platinum lesion (GG Pt and GTG Pt) were treated with the indicated amounts of NTPs at room temperature (RT) or 37°C. Pane A. 8% urea polyacrylamide gel analysis of RNA products. Panel B. enlarged portion of the gel data in A containing RNA transcripts of RNA EC62 and stalled elongation complexes at platinum damage sites. Panel C. sequence of the template strand up to the damage site (bottom lines) and of the RNA transcripts generated (top three lines). Reprinted with the kind permission of Dr. Stephen J. Lippard, MIT, and the American Society for Biochemistry and Molecular Biology from Lippard and Jung (2003) J. Biol. Chem. 278, 52084. |
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