Focus: Construction of an artificial elongation complex

Use of MagneHis™ Particles and His-Tagged RNA Polymerase to Study Stalled Elongation Complexes at DNA Lesions 

From the article Jung, Y. and Lippard, S.J. (2003) J. Biol. Chem. 278, 52084–92.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA

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

1. Yip, T.T., Nakagawa, Y. and Porath, J. (1989) Anal. Biochem. 183, 159–71.
2. Hutchens, T.W. and Yip, T.T. (1990) J. Chromatogr. 500, 531–42.
   

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