FIG. 3.

Stalled-transcript elongation and EMSA of ternary complexes. (A) Ternary complexes that contained the ³²P-labeled 24-nt U-less transcript were incubated without (−) or with HMtA2 and then added to a complete transcription reaction mixture and incubated at 58°C for 20 min. A control aliquot of the ternary complexes was incubated in a reaction mixture that lacked UTP (−UTP). The transcripts synthesized were separated by electrophoresis, and the ³²P-labeled transcripts were detected by autoradiography and quantitated by β-decay measurement. The HMtA2 dimer/100-bp ratio in the reaction mixture is indicated above the corresponding lane. The control lanes contained size standards (S1 and S2). (B) Aliquots of ternary complexes that contained a ³²P-labeled 24-nt U-less transcript were incubated with HMtA2 at the histone dimer/100-bp ratio indicated above each lane. The products were separated by electrophoresis and visualized by autoradiography. Control lanes contained a sample of ³²P-end-labeled template DNA (T), an aliquot of the ternary complexes incubated without HMtA2 addition (−), and an aliquot incubated with the HMfB K13T+R19S+T54K variant which lacks DNA binding ability (51) at a ratio of 30 histone dimers per 100 bp (C).