Differential Stability of Baculovirus Late Transcription Complexes during Initiation and Elongation

AbstractAnin vitrosystem for baculovirus late transcription was developed that allows comparison of conditions that affect transcription initiation and elongation. A series of synthetic promoters was constructed based on the baculovirus late p6.9 promoter. The modified promoters were designed with a cytidine-free region downstream of the late promoter in order to yield paused transcripts of defined lengths in the absence of CTP. Transcription was found to be more efficient from a supercoiled template than from a linear template for this promoter. The stalled transcription complex remained competent and could be elongated in the presence of a full set of nucleotides. This made it possible to separately test the effects of heat treatment and inhibition by sarkosyl and heparin on initiation and elongation. Elongation complexes were more resistent than initiation complexes to each of these treatments. Furthermore a 1–2 mM MgCl2concentration is critical for optimal initiation, but elongation can proceed in the presence of MgCl2concentrations as high as 20 mM