Template Strand Switching by T7 RNA Polymerase

Minqing Rong,Russell K Durbin,William T Mcallister

doi:10.1074/jbc.273.17.10253

Minqing Rong, Russell K Durbin + Show 1 more

Open Access

https://doi.org/10.1074/jbc.273.17.10253

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Abstract

T7 RNA polymerase (RNAP) is able to traverse a variety of discontinuities in the template (T) strand of duplex DNA, including nicks, gaps, and branched junctions in which the 3' end of the T strand is not complementary to the non-template (NT) strand. The products represent a faithful copy of the T strand, with no insertions or deletions. On double-stranded templates having protruding 3' ends the polymerase is able to insert the free 3' end of the NT strand and to utilize this as a new T strand ("turn around transcription"), resulting in the anomalous production of high molecular weight transcripts. The capacity of T7 RNAP to bypass interruptions in the T strand depends upon the stability of the elongation complex. Sequences that are expected to stabilize a local RNA:DNA hybrid (such as the presence of a C6 tract in the T strand) dramatically reduce dissociation of the RNAP while still allowing the enzyme to insert a new 3' end. Similar effects on RNAP release are observed when the enzyme reaches the end of a template (i.e. when synthesizing runoff products), resulting in markedly different yields of RNA product during multiple rounds of transcription.

Highlights

The capacity of T7 RNA polymerase (RNAP) to bypass interruptions in the T strand depends upon the stability of the elongation complex
In agreement with previous reports [16, 17] we found that T7 RNAP is able to traverse a gap of 1–2 nt in the template strand (Fig. 1B, lanes 5, 6, and 8), resulting in the synthesis of RNA products that correspond to a full-length transcript (47 nt) minus the interval defined by the gap (1 nt in lanes 5 and 8 and 2 nt in lane 6)
By using synthetic oligomers to generate a discontinuity in the T strand, we found that T7 RNAP is able to traverse a nick with an efficiency of jumping that is comparable to the efficiency of gap jumping (Fig. 1B, compare lane 7 with lanes 5, 6, and 8)

Summary

EXPERIMENTAL PROCEDURES

DNA Templates and RNA Polymerase—DNA oligomers were synthesized by Macromolecular Resources (Colorado State University) and purified by low pressure reverse phase chromatography. Transcription Assays—Unless otherwise noted, transcription was carried out in a volume of 10 ␮l in GHT buffer (see above) containing 0.5 mM ATP, CTP, GTP, and UTP (Amersham Pharmacia Biotech Ultrapure); 2 ␮Ci of [␣-32P]ATP or GTP (specific activity of 800 Ci/mmol; NEN Life Science Products); 10 –20 nM RNA polymerase, and 50 nM synthetic DNA or 1 ␮g of plasmid DNA as template. Reactions were incubated at 37 °C for 10 min and terminated by the addition of 10 ␮l of stop buffer, and the products were resolved by electrophoresis in polyacrylamide gels containing 7 M urea [21]. RNA was precipitated at Ϫ70 °C in the presence of 70% isopropyl alcohol and 0.3 M sodium acetate and resolved by electrophoresis in a 20% polyacrylamide gel as described above. The resulting products were digested with EcoRI and BamHI and cloned into the corresponding sites of pBluescript II SK(ϩ) (Stratagene); the DNA sequence of the cloned interval was determined using chain terminating dNTPs [22]

RESULTS

TABLE I Sequences of DNA oligomers

DISCUSSION