Deletion mapping of DNA regions required for SV40 early region promoter function in vivo.

Abstract

The SV40 early region promoter, previously localized to the DNA segment bounded by the HpaII and HindIII restriction sites (nucleotides 346 and 5171), was further defined by construction of an extensive set of deletions within this region and measurement of their effects on (a) viral DNA replication, (b) virus multiplication and the ability to complement early and late mutations, (c) transformation of rat cells, (d) large T antigen formation, and (e) the location of the 5' ends of early mRNAs. One set of mutations is represented by deletions that begin at the HpaII site and extend unidirectionally for varying lengths toward the BglI site at ori. A second set of mutants contains deletions that start at ori and extend unidirectionally for varying lengths towards the HpaII site. A third set of mutants, with deletions or duplications of various lengths and boundaries, lie between the HpaII and BglI sites. Our studies indicate the following. (a) Ori, the sequence needed for initiating SV40 DNA replication, extends from the sequences needed for initiating SV40 DNA replication, extends from the sequences needed to bind T antigen to the palindrome in site II to nucleotide 34, the late region edge of the AT block. Flanking sequences adjacent to the AT block facilitate DNA replication. (b) The SV40 early region promoter comprises two functionally distinct nucleotide sequence elements. One is flanked by nucleotides 5231 and 107, and contains the RNA initiation sites at nucleotides 5231-5237, a positioning element resembling the TATAAATA consensus sequence about 20-25 nucleotides upstream, and an RNA polymerase II recognition sequence contributed by short GC-rich sequences clustered between nucleotides 35 and 107; we refer to this as the RNA polymerase II interaction site. The second distinct sequence element is contained within each of two 72-bp segments located between nucleotides 107 and 250; the behavior of this element suggests that it may influence the accessibility of RNA polymerase II for the interaction site or the efficiency of RNA chain initiation. Large T antigen binding sites I, II, and III overlap with the putative RNA polymerase II interaction site; since large T antigen does not prevent elongation of RNA transcripts initiated upstream, T antigen probably represses early region expression by preventing RNA polymerase II binding to the promoter.