Elongation by Escherichia coli RNA polymerase is blocked in vitro by a site-specific DNA binding protein.
As a means of determining how elongating RNA polymerase responds to a protein in its path, transcription has been carried out in vitro with the purified Escherichia coli enzyme on templates associated with a sequence-specific DNA binding protein. The major RNA species generated is the length expected from RNA polymerase which has transcribed to the position of the bound protein and is unable to elongate further. The binding proteins used are two mutants of the EcoRI endonuclease which are defective in cleavage function but retain high affinity for the wild-type recognition sequence (Wright, D. J., King, K., and Modrich, P. (1989) J. Biol. Chem. 264, 11816-11821). Blockage of RNA polymerase occurs on linear and circular templates and, although efficient with both proteins, is more effective for the EcoRI derivative with the slower dissociation rate. The protein-blocked transcription complexes are stable over time and remain in an active form, resuming elongation when the blocking protein is displaced by an increase in ionic strength. These paused ternary complexes, if treated with the termination factor rho, undergo release. The 3' ends of the blocked-length RNAs from DNAs of distinct sequences reveal that the ternary complexes are positioned at a constant distance from the protein block, 14 nucleotides upstream of the EcoRI recognition sequence. This information is combined with exonuclease III footprinting data to position the 3' end of the nascent RNA chain in the ternary complex quite near (approximately 7 nucleotides) the leading edge of RNA polymerase.
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