Figure 4 - Antibodies to -actin and NEM-modified S1 inhibit transcription in a purified system.

(a) Schematic representation of the transcription assay. Recombinant transcription factors TBP, TFIIF, TFIIB and purified RNA polymerase II were incubated with 2 g each of the indicated antibodies or 7 g of NEM-modified S1 (NEM-S1) for 20 min. The AdMLP template was added and the reaction mixture was incubated for another 10 min. The transcription reaction was then initiated by adding NTPs. The reaction was terminated as required and the transcripts were purified and analysed as described in the Methods section. (b) The effect of actin antibodies and NEM-S1 on transcription. The same actin antibodies used in Fig. 3a were incubated with an aliquot of the purified RNA polymerase II²² and assayed as shown in a. Note that the antibodies that recognize actin in Fig. 3a (C4, 56-4 and anti -actin) inhibit transcription. In contrast, antibodies to muscle actin that do not recognize actin in Fig. 3a (B4 and HUC 1-1) had no effect on transcription. NEM-S1, which binds specifically and tightly to actin²³, also inhibited transcription. To demonstrate the specificity of this effect, 2 g of the -actin antibody (which gives maximal inhibition) was pre-incubated with 0.5 g of purified -actin or buffer on ice for 30 min before incubation with the purified RNA polymerase II and recombinant transcription factors. The purified -actin prevented the -actin antibody from inhibiting transcription (last lane). The control represents samples incubated with buffer only. Each experiment was repeated 3–5 times. (c) Adding -actin stimulates transcription by RNA polymerase II. HPLC-purified RNA polymerase II²⁴ was incubated with varying amounts of purified -actin for 30 mins on ice before initiation of the transcription assay (a). Note that 4 ng of exogenous -actin stimulates transcript accumulation by almost eightfold and that there is no further increase in the presence of 10 ng of -actin.