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J Biol Chem, Vol. 273, Issue 17, 10253-10260, April 24, 1998

Template Strand Switching by T7 RNA Polymerase

From the Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York, Health Science Center, Brooklyn, New York 11203-2098

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.

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