RNA displacement pathways during transcription from synthetic RNA-DNA bubble duplexes.
Previously [Daube, S.S., & von Hippel, P.H. (1992) Science 258, 1320] we have shown that functional transcription elongation complexes can be formed by adding ribonucleotide triphosphates, Mg2+, and either Escherichia coli or T7 RNA polymerase to synthetic RNA-DNA bubble-duplex constructs. Here these observations are extended to show that the RNA transcripts synthesized from these bubble-duplex constructs are properly displaced from the DNA template during transcription. Some details of the displacement process differ between the polymerases tested. Thus the transcript is fully and processively displaced in the course of T7 polymerase-catalyzed synthesis from the bubble-duplex constructs, while the presence of a large excess of an RNA (or DNA) oligomer complementary to the DNA template sequence within the "permanent" DNA bubble is required to attain complete displacement of the nascent RNA from the construct during synthesis with the core E. coli enzyme. In addition, a correlation is shown between proper RNA displacement and the achievement of full-length transcript synthesis. We conclude that both the T7 polymerase and the E. coli core enzyme actively displace the nascent transcript during elongation and that the requirement for an RNA trap with the E. coli enzyme reflects its slower rate of synthesis. This suggests that these experiments may provide insight into the relative rates of transcript elongation and secondary structure formation within the nascent RNA in elongation and termination. By use of the RNA oligomer trap methodology, multiple rounds of transcript synthesis should be achievable on these bubble-duplex constructs with any polymerase.
Daube
S S
SS
Department of Chemistry, University of Oregon, Eugene 97403.
von Hippel
P H
PH
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008961:Models, Structural; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D006163:Ribonuclease T1; D014158:Transcription, Genetic
D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D006163:Ribonuclease T1