PAUSING AND TERMINATION BY BACTERIOPHAGE-T7 RNA-POLYMERASE

Two types of sites are known to cause pausing and/or termination by ba cteriophage T7 RNA polymerase (RNAP). Termination at class I sites (ty pified by the signal found in the late region of T7 DNA, T Phi) involv es the formation of a stable stem-loop structure in the nascent RNA ah ead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the clo ned human preproparathyroid hormone (PTH) gene, generate no evident st ructure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I an d class II sites may involve non-equivalent mechanisms, as mutants of T7 RNA polymerase have been identified that fail to recognize class II sites yet continue to recognize class I sites. In this work, we have analyzed pausing and termination at several class II sites, and varian ts of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in th e non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the pres ence of runs of Il, perhaps because they destabilize an RNA:DNA hybrid . The effects of T7 lysozyme on pausing and termination are consistent with the idea that termination involves a reversion of the polymerase from the elongation to the initiation conformation, and that lysozyme inhibits the return to the elongation conformation. A kinetic model o f pausing and termination is presented that provides a consistent inte rpretation of our results. (C) 1998 Academic Press.