Intrinsically bent DNA in a eukaryotic transcription factor recognition sequence potentiates transcription activation.
Many eukaryotic transcription factors induce DNA bending on binding to their recognition sequences. DNA bending could play a structural role by altering contacts between the protein and DNA. Alternatively, DNA bending could play a more direct role in transcription activation. To distinguish between these possibilities, we inserted two to eight copies of the intrinsic bending sequence, AAAAAACGTG, into a minimal promoter containing only a TATA box. The intrinsic DNA bending sequence was a potent activator of transcription in both in vivo transfection experiments and in a cell-free transcription system. A protein binds to the intrinsic bending sequence with high specificity in gel mobility shift assays and was required for its transcription in cell-free extracts. The intercalator, distamycin, which eliminates the ability of the sequence to bend, specifically reduced its transcription by about 60%. Mutations in the sequence which abolished DNA bending reduced transcription by approximately 70% in vivo. Competition gel mobility shift assays showed that the transcription factor bound equally well to mutants in which DNA bending was abolished and to the intrinsic bending sequence. These data indicate that DNA bending can play a direct role in the activation of eukaryotic transcription.
Department of Biochemistry, University of Illinois, Urbana 61801.
J
J
Kim
S
S
Klooster
D J
DJ
Shapiro
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002474:Cell-Free System; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004214:Distamycins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D004214:Distamycins; D014157:Transcription Factors; C030000:stallimycin; D004247:DNA