Analysis of the in vitro synthesis of 5'-gamma-32P-labeled transcripts from coliphage lambda by gel electrophoresis, RNA-DNA hybridization, and RNase T1 digestion.

Abstract

Phage lambda RNA was synthesized in vitro in the presence of [gamma-32P]ATP or GTP. The 12 S, 9 S, 8 S, 6 S, and 5 S transcripts were labeled specifically at the 5'-end with [gamma-32P]ATP, while [gamma-32P]GTP was incorporated at the 5'-end of the 4 S RNA. Transcription of lambda DNA at various salt concentrations was studied by three experimental methods: electrophoresis of the transcripts in polyacrylamide gels; hybridization of RNA to DNA restriction fragments transferred to nitrocellulose paper; and digestion of the transcripts with RNase T1 and analysis of the resulting 5'-end 32P-labeled oligonucleotides by 20% polyacrylamide gel electrophoresis. Transcription from promoters pL, pR, and p'R was maximal between 50 mM and 150 mM NaCl and diminished as the salt concentration was raised to 200 mM. RNA synthesis originating from p'R was predominant at all salt concentrations. The relative salt sensitivity of RNA synthesis was determined for these promoters and found to decrease in the order: pL > pR > p'R.