Deoxyribonucleic acid-directed in vitro synthesis of ilv-specific messenger ribonucleic acid by extracts of Escherichia coli K-12.
The synthesis of ilv-specific messenger ribonucleic acid (mRNA) by extracts of Escherichia coli K-12 has been demonstrated in a deoxyribonucleic acid (DNA)-dependent, coupled transcription-translation system. ilv-Specific mRNA was determined by hybridization either to double-stranded lambdacI857St68h80dilv DNA (lambdah80dilv DNA) immobilized on nitrocellulose filters or to its separate l and r strands in liquid. During conditions optimal for protein synthesis, slightly more than 6% of the total [(3)H]RNA synthesized by S-30 extracts of the threonine deaminase-negative strain CU5136 was ilv-specific. Of this RNA, nearly 30% was complementary to the l (correct) strand. Total ilv-specific mRNA synthesis in vitro was not affected by omission of valine or all 20 amino acids from the reaction mixture. Hybridization of ilv-specific mRNA made in vitro to the l strand of lambdah80dilv DNA was effectively reduced in the presence of unlabeled RNA extracted from an ilv derepressed strain but not from an ilv deletion strain. In a purified transcription system, employing commercial RNA polymerase, twofold more ilv-specific mRNA was synthesized than in the coupled system, but this increase was entirely due to greater transcription of the r (incorrect) strand. An S-30 extract prepared from a strain isogenic to strain CU5136 but derepressed for ilvA gene expression synthesized twofold more ilv-specific mRNA in the coupled system. The significance of these findings is discussed.
D001426:Bacterial Proteins / Q000096:biosynthesis; D002250:Carbon Radioisotopes; D002458:Cell Fractionation; D002474:Cell-Free System; D003090:Coliphages; D003597:Cytosine Nucleotides / Q000378:metabolism; D004267:DNA Viruses; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D005796:Genes; D006836:Hydro-Lyases / Q000096:biosynthesis; D009154:Mutation; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis*; D013698:Templates, Genetic; D013912:Threonine; D014158:Transcription, Genetic; D014316:Tritium; D014500:Uracil Nucleotides / Q000378:metabolism; D014633:Valine / Q000378:metabolism
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D003597:Cytosine Nucleotides; D004279:DNA, Viral; D012329:RNA, Bacterial; D012333:RNA, Messenger; D014500:Uracil Nucleotides; D014316:Tritium; D013912:Threonine; D012321:DNA-Directed RNA Polymerases; D006836:Hydro-Lyases; D014633:Valine