Comparison of the polyoma virus early and late promoters by transcription in vitro

Parmjit Jat,Jeffrey W Roberts,Alison Cowie,Robert Kamen

doi:10.1093/nar/10.3.871

Abstract

Polyoma virus DNA was transcribed in the HeLa whole cell extract in vitro system (1). Early region transcripts with the same 5'-ends as in vivo mRNAs, located 31 +/- 2bp from 'TATA'-boxes, were synthesized by RNA polymerase II. Sequences sufficient for efficient expression of the early promoter were present in a substitution mutant lacking viral DNA from a position 55bp before the principal cap sites. Late region transcripts were synthesised inefficiently. Only one (at nt5129 +/- 2) of the many late mRNA cap sites functioned as an in vitro initiation point. This was the one 5'-end located 31 +/- 2bp from a sequence resembling the 'TATA' consensus. The proportion of late to early region RNA polymerase II transcripts decreased dramatically at suboptimal template concentrations. An hypothesis to explain the regulation of late gene expression in vivo based on these results is proposed. A linear templates were transcribed only by RNA polymerase II, transcripts with the same sense as late mRNAs and 5'-ends at nt5076 +/- 2 were produced from superhelical template by an alpha amanitin resistant enzyme.

Highlights

The development of 1n vitro systems [1,2] which faithfully Initiate the transcription of eucaryotic genes has facilitated analysis of the DNA sequence elements comprising transcriptional promoters 1n higher organisms
Transcription of viral DNA cut only at a site in the early region resulted in the selective disappearance of the 500 nucleotide RNA, suggesting that i t alone resulted from transcription towards the l a t e region (Figure 2A, track " E " )
We present elsewhere evidence directly demonstrating the principal for Py early region transcripts (A.C. ^t ^1_, submitted)

Summary

Introduction

The development of 1n vitro systems [1,2] which faithfully Initiate the transcription of eucaryotic genes has facilitated analysis of the DNA sequence elements comprising transcriptional promoters 1n higher organisms (reviewed 1n refs. 3-4). We have been studying the transcription of polyoma virus (Py) DNA 1n vivo for a number of years. The genetic structure of Py DNA 1s well understood [5], and the viral RNAs synthesized during productive infection and 1n transformed cell lines have been characterized in molecular detail [5,6,7,8,9]. The viral DNA has two transcription units, the early and the late, extending In opposite directions from near the origin of DNA replication around the circular genome (Figure 1 ). The early transcription unit 1s expressed throughout lytic Infection and in transformed cells. At late times of productive Infection, Its activity 1s negatively regulated by one of Its gene products, the large T-prote1n [7,10].

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Conclusion