TY - JOUR
T1 - Intragenic activating and repressing elements control transcription from the adenovirus IVa2 initiator
AU - Chen, Hangjiong
AU - Vinnakota, R.
AU - Flint, S. J.
PY - 1994
Y1 - 1994
N2 - The downstream stimulatory segment of the adenovirus type 2 IVa2 promoter includes a TA-rich sequence that binds recombinant TATA-binding proteins (TBP) in vitro. We now demonstrate that when placed upstream of the IVa2 initiator, this TA-rich sequence operated as a TATA element but exhibited significantly lower transcriptional and TBP-binding activities than did the TATA box of the adenovirus major late (ML) promoter. In sharp contrast, changing the IVa2 TA-rich sequence in its natural, intragenic context to the ML TATA sequence increased the activity of the IVa2 promoter only slightly. In view of this discrepancy, we examined the effects of single, double, and clustered point mutations in the downstream sequence on the activity of a minimal IVa2 promoter. Mutations between positions +21 and +29 inhibited IVa2 transcription, in some cases to the very low level directed by the IVa2 initiator alone. By contrast, substitutions within the TA-rich sequence increased the efficiency of IVa2 transcription. These results indicated that the downstream, TA-rich sequence does not function as an intragenic TFIID-binding site but rather is included within a negative regulatory element. Electrophoretic mobility shift and methylation interference assays using wild-type and mutated, intragenic promoter sequences identified a HeLa cell component whose binding to the sequence +11 to +27 correlated with repression of IVa2 transcription, suggesting that a negative regulatory element is superimposed upon the intragenic sequence required for efficient transcription from the IVa2 initiator.
AB - The downstream stimulatory segment of the adenovirus type 2 IVa2 promoter includes a TA-rich sequence that binds recombinant TATA-binding proteins (TBP) in vitro. We now demonstrate that when placed upstream of the IVa2 initiator, this TA-rich sequence operated as a TATA element but exhibited significantly lower transcriptional and TBP-binding activities than did the TATA box of the adenovirus major late (ML) promoter. In sharp contrast, changing the IVa2 TA-rich sequence in its natural, intragenic context to the ML TATA sequence increased the activity of the IVa2 promoter only slightly. In view of this discrepancy, we examined the effects of single, double, and clustered point mutations in the downstream sequence on the activity of a minimal IVa2 promoter. Mutations between positions +21 and +29 inhibited IVa2 transcription, in some cases to the very low level directed by the IVa2 initiator alone. By contrast, substitutions within the TA-rich sequence increased the efficiency of IVa2 transcription. These results indicated that the downstream, TA-rich sequence does not function as an intragenic TFIID-binding site but rather is included within a negative regulatory element. Electrophoretic mobility shift and methylation interference assays using wild-type and mutated, intragenic promoter sequences identified a HeLa cell component whose binding to the sequence +11 to +27 correlated with repression of IVa2 transcription, suggesting that a negative regulatory element is superimposed upon the intragenic sequence required for efficient transcription from the IVa2 initiator.
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U2 - 10.1128/MCB.14.1.676
DO - 10.1128/MCB.14.1.676
M3 - Article
C2 - 8264636
AN - SCOPUS:0027976324
SN - 0270-7306
VL - 14
SP - 676
EP - 685
JO - Molecular and cellular biology
JF - Molecular and cellular biology
IS - 1
ER -