DIFFERENTIAL STABILITY OF BACULOVIRUS LATE TRANSCRIPTION COMPLEXES DURING INITIATION AND ELONGATION

An in vitro system for baculovirus late transcription was developed th at allows comparison of conditions that affect transcription initiatio n and elongation. A series of synthetic promoters was constructed base d on the baculovirus late p6.9 promoter. The modified promoters were d esigned with a cytidine-free region downstream of the late promoter in order to yield paused transcripts of defined lengths in the absence o f CTP. Transcription was found to be more efficient from a supercoiled template than from a linear template for this promoter. The stalled t ranscription complex remained competent and could be elongated in the presence of a full set of nucleotides. This made it possible to separa tely 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. Furth ermore a 1-2 mM MgCl2 concentration is critical for optimal initiation , but elongation can proceed in the presence of MgCl2 concentrations a s high as 20 mM. (C) 1998 Academic Press.