Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism.
A recent model for the mechanism of intrinsic transcription termination involves dissociation of the RNA from forward-translocated (hypertranslocated) states of the complex [Yarnell WS, Roberts JW (1999) Science, 284:611-615]. The current study demonstrates that halted elongation complexes of T7 RNA polymerase in the absence of termination signals can also dissociate via a forward-translocation mechanism. Shortening of the downstream DNA or the introduction of a stretch of mismatched DNA immediately downstream of the halt site reduces a barrier to forward translocation and correspondingly reduces the lifetime of halted complexes. Conversely, introduction of a cross-link downstream of the halt site increases the same barrier and leads to an increase in complex lifetime. Introduction of a mismatch within the bubble reduces a driving force for forward translocation and correspondingly increases the lifetime of the complex, but only for mismatches at the upstream edge of the bubble, as predicted by the model. Mismatching only the two most upstream of the eight bases in the bubble provides a maximal increase in complex stability, suggesting that dissociation occurs primarily from early forward-translocated states. Finally, addition in trans of an oligonucleotide complementary to the nascent RNA just beyond the hybrid complements the loss of driving force derived from placement of a mismatch within the bubble, confirming the expected additivity of effects. Thus, forward translocation is likely a general mechanism for dissociation of elongation complexes, both in the presence and absence of intrinsic termination signals.
Zhou
Yi
Y
Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003, USA.
Navaroli
Deanna M
DM
Enuameh
Metewo Selase
MS
Martin
Craig T
CT
D001483:Base Sequence; D001692:Biological Transport; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases