Mouse‐Myeloma RNA Polymerase B Template Specificities and the Role of a Transcription‐Stimulating Factor

A DNA‐dependent transcriptase has been purified from mouse myeloma tissue. The system consists of an RNA polymerase of type B and a basic, heat‐labile protein of M r 20000–30000 (stimulating factor). The RNA polymerase was characterized by its requirement for Mn 2+ and medium ionic strength as well as by its susceptibility to inhibition by α‐amanitin and phosphate ions. The template specificities were studied using native and heat‐denatured DNA as well as synthetic double and single‐stranded polydeoxynucleotides. Alternating polydeoxynucleotides like poly[d(A‐T) · d(A‐T)] and poly[d(I‐C) · d(I‐C)] are excellent templates, the latter exceeding native DNA by two orders of magnitude. With these templates, factor stimulation is less expressed and at most two‐fold. With DNA‐like templates, such as poly[d(A‐C) · d(T‐G)] and poly[d(A‐G) · d(T‐C)], transcription proceeds at a lower rate (though still exceeding native DNA), but factor can stimulate up to 5‐fold. In contrast, the homoduplexes poly(dA) · poly(dT), poly(dG) · poly(dC), and poly(dI) · poly(dC) are transcribed poorly, if at all, irrespective of the presence or absence of stimulating factor. Among the single‐stranded polydeoxynucleotides tested poly[d(A‐C)], poly(dA), poly(dI), and poly(dC) are moderately well transcribed, whereas poly[d(T‐G)] and poly(dI) are completely ineffective. Initiation of transcription was studied using [γ‐ 32 P]ATP. From the results it is concluded that the stimulating factor is involved in the progress of transcription rather than in initiation.