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J. Biol. Chem., Vol. 269, Issue 48, 30101-30104, 12, 1994
G Pan and J Greenblatt
To further elucidate the mechanism of transcriptional initiation, we used
synthetic oligonucleotides to prepare templates containing heteroduplex
regions of varying size and location along the DNA of the adenovirus major
late promoter. Unlike closed, linear DNA, or DNA with a downstream
mismatch, DNA with a mismatch upstream of the initiation site only required
the general factors TATA box-binding protein and transcription factor (TF)
IIB to direct specific and accurate initiation in vitro by calf thymus RNA
polymerase II. In the presence of TFIIF, initiation was possible on closed,
linear DNA, but an upstream mismatch region still stimulated
transcriptional initiation by more than 100-fold, leading to production of
approximately 0.5 transcript/template in the absence of TFIIE, TFIIH, or
ATP. The presence of a DNA mismatch was most effective in the -9 to -1
region; furthermore, stimulation by a mismatch did not require that the
initiation site be included in the heteroduplex region. Efficient
initiation at the immunoglobulin heavy chain promoter in the presence of
TATA box-binding protein and TFIIB was also achieved when a mismatch region
was introduced from -9 to +3. Our results suggest that initiation by RNA
polymerase II in the absence of transcriptional activation is limited by
melting of the promoter DNA upstream of the initiation site.
Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site
Banting and Best Department of Medical Research, University of Toronto, Canada.
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