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.