Selectivity of RNA chain initiation in vitro. 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 lambdacb2 and T7. Determination of synthesis from specific promoters is carried out by the fractionation and quantitation on polyethylenimine-cellulose thin-layer chromatograms of the 5'-terminal oligonucleotides produced by digestion of the RNA products with T1 RNase. The major findings of this work are that (1) lambdacb2 promoters are more salt sensitive than T7 promoters and the salt concentration affects individual promoters differently, (2) T7 promoters initiate maximally at 37 degrees 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 lambdacb2 promoters show differences in stability when challenged with heparin.