Experimentally determined weight matrix definitions of the initiator and TBP binding site elements of promoters.

The basal elements of class II promoters are: (i) a-30 region, recognized by TATA binding protein (TBP); (ii) an initiator (Inr) surrounding the start site for transcription; (iii) frequently a downstream (+10 to +35) element. To determine the sequences that specify an Inr, we performed a saturation mutagenesis of the Inr of the SV40 major late promoter (SV40-MLP). The transcriptional activity of each mutant was determined both in vivo and in vitro. An excellent correlation between transcriptional activity and closeness of fit to the optimal Inr sequence, 5'-CAG/TT-3', was found to exist both in vivo and in vitro. Employing a neural network technique we generated from these data a weight matrix definition of an Inr that can be used to predict the activity of a given sequence as an Inr. Using saturation mutagenesis data of TBP binding sites we likewise generated a weight matrix definition of the -30 region element. We conclude the following: (i) Inrs are defined by the nucleotides immediately surrounding the transcriptional start site; (ii) most, if not all, Inrs are recognized by the same general transcription factor(s). We propose that the mechanism of transcription initiation is fundamentally conserved, with the formation of pre-initiation complexes involving the concurrent binding of general transcription factors to the -30, Inr and, possibly, downstream elements of class II promoters.

SV40 early-to-late switch involves titration of cellular transcriptional repressors.

We have purified factors from HeLa cell nuclear extracts that bind to the transcriptional initiation site of the SV40 major late promoter (SV40-MLP). The resulting fraction consists predominantly of three proteins, collectively called initiator-binding protein of SV40 (IBP-s) with electrophoretic mobilities of approximately 45-55 kD. Gel mobility-shift and DNase I-protection analyses indicate that each of these three proteins associates with high affinity to sequences located at the initiation site and 55 bp downstream of it. IBP-s-binding sites with lower affinities are located at +5 and +30. Addition of purified IBP-s to a cell-free transcription system represses transcription from the SV40-MLP, but not the SV40 early promoter. SV40 mutants lacking the two strongest IBP-s-binding sites (1) are not repressed by the addition of IBP-s in vitro, (2) overproduce late RNA (relative to wild-type SV40) at low, but not high, template copy number in vitro, and (3) exhibit increased levels of late RNA at early, but not late, times after transfection into CV-1 cells. Therefore, IBP-s is a cellular repressor of transcription of the SV40-MLP that may, in large part, be responsible for the replication-dependent component of the early-to-late shift in SV40 gene expression. Partial amino acid sequence data obtained from the approximately 55-kD component of IBP-s indicate that it is hERR1, an orphan member of the steroid-thyroid hormone receptor superfamily. These findings suggest simple molecular mechanisms by which hormones may modulate expression of viral late genes. We speculate that activation of expression of the late genes of other viruses may occur by similar mechanisms.