Role of the 21,000 molecular weight polypeptide of Bacillus subtilis RNA polymerase in RNA synthesis.

DNA-dependent RNA polymerase from Bacillus subtilis contains a 21,000 molecular weight (21K) peptide subunit. This subunit was purified and added to 21K-depleted polymerase isolated from both uninfected and SP85-infected B. subtilis. The effect of the subunit on total RNA synthesis, on enzyme-DNA binding, on RNA chain initiation and elongation, and on enzymatic specificity were examined. A comparison was made of the effects of the 21K peptide and NaCl on polymerase activity, RNA chain elongation, and symmetry of transcription of SP82 DNA. The addition of the 21K peptide to enzyme preparations lacking this subunit stimulated total polymerase activity 20 to 40%. In contrast, addition of NaCl at concentrations greater than 0.1 M significantly reduced polymerase activity. The 21K peptide appeared to alter the general affinity of the polymerase for DNA. The rate of RNA chain initiation was not affected by the 21K peptide, but RNA chain elongation was stimulated. Both the 21K peptide and NaCl increased the asymmetry of transcription of SP82 DNA by phage-modified polymerase. The 21K effect was related to the stimulation of elongation while high concentrations of NCl appeared to act at RNA chain initiation. RNAs synthesized in vitro by polymerase lacking and supplemented with the 21K peptide were translated by a Escherichia coli cell-free system. The 21K peptide had little direct effect on the selection of promoters in vitro as measured by this technique, but it dramatically increased the translatability of the product.