Coupling of RNA displacement and intrinsic termination in transcription from synthetic RNA.DNA bubble duplex constructs.
Functional transcription elongation complexes can be formed by adding RNA polymerase in trans to a preformed nucleic acid construct. This construct consists of a double-stranded DNA fragment that contains a noncomplementary (permanent DNA bubble) region into which an RNA primer oligonucleotide has been hybridized. By ligating a DNA fragment containing the strong intrinsic terminator T7Te to the RNA.DNA bubble duplex, we show here that Escherichia coli core RNA polymerase-catalyzed transcription, initiated from such a construct, terminates at the predicted position. Furthermore, we show that the termination efficiency obtained is comparable to that observed in a control reaction initiated with the E. coli holopolymerase from the T7A1 promoter if an RNA oligomer trap is used to permit proper displacement of the nascent RNA from the DNA template strand. The trap oligomer is complementary to the template strand of the permanent DNA bubble and prevents rehybridization of the nascent RNA at this site. Varying the amount of RNA trap that is added permits us to modulate the extent of total RNA displacement. Our results show that RNA displacement and termination efficiency are directly correlated, suggesting that intrinsic termination requires that the nascent RNA be free to assume its solution conformation. Several models of intrinsic termination are presented and discussed in light of these data.
Daube
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SS
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Hart
C R
CR
von Hippel
P H
PH
D017123:Bacteriophage T7 / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI; D004926:Escherichia coli / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D009843:Oligoribonucleotides; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D015245:Deoxyribonuclease BamHI