TY - JOUR
T1 - Pausing and termination by bacteriophage T7 RNA polymerase
AU - Lyakhov, Dmitry L.
AU - He, Biao
AU - Zhang, Xing
AU - Studier, F. William
AU - Dunn, John J.
AU - McAllister, William T.
N1 - Funding Information:
Work at SUNY Health Science Center at Brooklyn was supported by NIH grant GM38147 to W.T.M. Work at Brookhaven National Laboratory was supported by the Office of Biological and Environmental Research of the US Department of Energy. We are grateful to Mr Ray Castagna for technical assistance, and to Ms Rita Gould and Ms Roseann Lingeza for secretarial assistance.
PY - 1998/7/10
Y1 - 1998/7/10
N2 - Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP). Termination at class I sites (typified by the signal found in the late region of T7 DNA, TΦ) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the cloned human pre-proparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I and 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 variants of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, 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 of pausing and termination is presented that provides a consistent interpretation of our results.
AB - Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP). Termination at class I sites (typified by the signal found in the late region of T7 DNA, TΦ) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the cloned human pre-proparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I and 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 variants of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, 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 of pausing and termination is presented that provides a consistent interpretation of our results.
KW - Conformation change
KW - T7 lysozyme
KW - Termination kinetics
KW - Transcript termination
KW - Transcriptional pausing
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U2 - 10.1006/jmbi.1998.1854
DO - 10.1006/jmbi.1998.1854
M3 - Article
C2 - 9654445
AN - SCOPUS:0032504089
SN - 0022-2836
VL - 280
SP - 201
EP - 213
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -