Mechanism of instability in abortive cycling by T7 RNA polymerase.
Abortive transcription, the premature release of short transcripts 2-8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies, results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a novel DNA construct containing a nick between positions -14 and -13 of the nontemplate strand suggests that the more flexible promoter reduces somewhat the strain inherent in initially transcribing complexes, with a resulting decrease in abortive product release. Finally, as assessed by exonuclease III footprinting and transcription profiles, a DNA construct defective in bubble collapse specifically from the downstream end exhibits less abortive cycling and little perturbation of the final transition to elongation, including the process of promoter release.
Gong
Peng
P
Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003-9336, USA.
Martin
Craig T
CT
D020413:3' Untranslated Regions / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics*; D020137:Base Pair Mismatch / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000821:virology*; D010441:Peptide Chain Elongation, Translational / Q000235:genetics; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D020413:3' Untranslated Regions; D004279:DNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases