title: Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase.
abstract: 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-template. 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 theoretically unable to form 3'-end base-paired duplexes. The full-length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions.
authors.affiliation: Instituto de Parasitología y Biomedicina López-Neyra CSIC, Ventanilla, Granada, Spain.
authors.forename: Genoveva A
authors.initials: GA
authors.lastname: Nacheva
authors.forename: Alfredo
authors.initials: A
authors.lastname: Berzal-Herranz
mesh_terms: D020029:Base Pairing; D002384:Catalysis; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D015723:Gene Library; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism; D018075:RNA, Complementary / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000502:physiology*; D014764:Viral Proteins
chemical_list: D016337:RNA, Catalytic; D018075:RNA, Complementary; D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases