SELECTIVITY OF RNA CHAIN INITIATION INVITRO .3. VARIABLES AFFECTING INITIATION OF TRANSCRIPTION

The effects of salt, temperature, enzyme to DNA ratio, and heparin challenge on both total RNA synthesis and synthesis from specific promoters are examined using DNA from bacteriophages λcb2 and T7. Determination of synthesis from specific promoters is carried out by the fractionation and quantitation on polyethylenimine-cellulose thin-layer chro-matograms of the 5′-terminal oligonucleotides produced by digestion of the RNA products with T1 RNase. The major findings of this work are that (1) λcb2 promoters are more salt sensitive than T7 promoters and the salt concentration affects individual promoters differently, (2) T7 promoters initiate maximally at 37 °C but the transition temperatures of promoters vary and may be dependent on the salt concentration, (3) increasing the enzyme to DNA ratio results in increasing initiations at the promoters on T7 DNA without causing measurable initiation at non-promoters, and (4) T7 and λcb2 promoters show differences in stability when challenged with heparin. © 1978, American Chemical Society. All rights reserved.