Premium
Discontinuous movement and conformational change during pausing and termination by T7 RNA polymerase
Author(s) -
Mukherjee Srabani,
Brieba Luis G.,
Sousa Rui
Publication year - 2003
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/cdg618
Subject(s) - biology , polymerase , processivity , biophysics , transcription (linguistics) , rna polymerase ii , conformational change , microbiology and biotechnology , termination factor , rna polymerase , rna , dna , biochemistry , rna dependent rna polymerase , gene , linguistics , philosophy , promoter , gene expression
Time‐resolved characterization of T7 RNA polymerase pausing and terminating at a class II termination site has been carried out using site‐specifically tethered chemical nucleases. The data indicate that T7RNAP normally moves uniformly down the template as a rigid body. However, at the class II site this movement is interrupted, and the leading edge of the polymerase moves further along the DNA than the trailing edge. This discontinuous movement may persist until it can no longer be accommodated by conformational changes in the elongation complex, at which point the polymerase can either pause or terminate. Termination, but not pausing, is abrogated by introduction of a disulfide bond between the polymerase fingers and thumb subdomains. The introduced cysteines disrupt a thumb–fingers salt‐bridge and, under reducing conditions, this mutant enzyme shows reduced processivity coincident with extension of the RNA to 5 nt. These observations suggest that termination requires that the thumb and fingers subdomains move apart, in a reversal of a conformational change important for initially forming a stable transcription complex.