Plasmids allowing transcription of cloned DNA by Salmonella typhimurium phage SP6 RNA polymerase to produce RNAs with authentic 5'-terminal sequences.
We wished to determine whether there is any specific sequence downstream of the start point of the SP6 promoter which is required for its function in the plasmid pSP64 (Melton et al., 1984). Lack of such specificity would permit in vitro synthesis of an RNA molecule having a 5'-terminal sequence identical to its wild-type in vivo counterpart. To test its requirement, we replaced all of the SP6 sequence downstream of the transcription start point with heterologous nucleotides (nt) and found that any sequence will suffice to permit efficient and accurate transcription. These results permitted construction of plasmids for synthesis of 'authentic' transcripts from cloned DNA. In one case, by an oligodeoxynucleotide-mediated site-specific deletion, we placed the start point of yeast gene TCM1 at nt + 2 of the SP6 promoter and produced in vitro TCM1 mRNA with a wild-type 5'-terminal sequence. We also constructed a vector, pSP64 delta 1, in which the SalI/AccI/HincII recognition sites of pSP64 reside at nt + 2 through + 7. Plasmids such as pSP64 delta 1 may be more useful in some cases as insertion of any DNA fragment at one of these three sites will yield a transcript in which only two to four nt are derived from the vector.
D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009876:Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D012483:Salmonella Phages / Q000201:enzymology / Q000235:genetics*; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*; D013045:Species Specificity; D014158:Transcription, Genetic
D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes