tailieunhanh - Báo cáo khoa học: Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase

The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotide-long sequences under the control of the minimal bac-teriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11. Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17-nucleotide-long RNA as the only detectable run-off product (only 15% of the total), and (b) templates that in addition to the expected full-length RNA,. | Eur. J. Biochem. 270 1458-1465 2003 FEBS 2003 doi Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase Genoveva A. Nacheva and Alfredo Berzal-Herranz Instituto de Parasitologia y Biomedicina Lopez-Neyra CSIC Ventanilla Granada Spain The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotide-long sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions 8 10 and 11. Two clearly distinct types of template were identified which produced the range of transcription patterns observed a those that yielded 17-nucleotide-long RNA as the only detectable run-off product only 15 of the total and b templates that in addition to the expected full-length RNA produced other products longer than 17 nucleotides. Self-complementarity analysis of the expected run-off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non-base-paired 3 -ends. However the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3 -end complementarity therefore yielding an RNA-primed RNA-tem-plate. The gel-purified 17-nucleotide-long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template. No extension was observed when assaying the 17-nucleotide RNA products resulting from the first type of template. We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA-primed template into a nonextendible dimer thus leading to a drastic switch of the 17-nucleotide product yield from less than 10 to 100 . Further two type B DNA templates were extended by two nucleotides at the 3 -end to produce RNA transcripts .