Abstract
The proximal or core promoter of a typical eukaryotic protein coding gene comprises distinct elements, TATA and/or initiator (Inr). The existence of TATA or Inr at the core promoter suggests that the mechanism of transcription initiation mediated by these two genetic elements may be different. Accordingly, it has been demonstrated that the transcriptional requirements for the TATA-containing, Inr-less (TATA+Inr-) promoters are different from the transcriptional requirements for the TATA-less, Inr-containing (TATA-Inr+) promoters. Although both types of promoters require the transcription initiation factor (TFIID) in addition to other common initiation factors, a TATA-Inr+ promoter requires accessory components. Here we have employed in vitro analyses to address the transcription factor requirements for a TATA-Inr+ promoter. We demonstrate that in addition to TFIID, a naturally occurring TATA-Inr+ promoter requires TFII-I, an Inr element-dependent transcription factor. Consistent with its Inr element-dependent activities, TFII-I is dispensable for a TATA+Inr- promoter. Furthermore, we demonstrate that both TFII-I and TFIID activities in nuclear extracts are temperature-sensitive. However, TFII-I is heat-inactivated at temperatures lower than that required to inactivate TFIID. Therefore, differential heat treatment of nuclear extracts provides an assay to discriminate between transcriptional requirements at TATA+Inr- and TATA-Inr+ promoters.
Highlights
Transcription initiation of protein coding genes is brought about by RNA polymerase II and a set of general transcription initiation factors [1,2,3]
The E-box containing oligonucleotide failed to abrogate Inr-dependent transcription. These results demonstrate that TFII-I binds to the V Inr element and that this binding is necessary for the V transcription initiation
Our analyses demonstrated that the TFII-I binding activity in a nuclear extract was abrogated by heat treatment of the nuclear extract minimally at 42 °C for 6 min
Summary
Transcription initiation of protein coding genes is brought about by RNA polymerase II and a set of general transcription initiation factors [1,2,3]. The TATA-binding transcription factor complex TFIID is required for both promoters [7], and heat treatment of nuclear extracts (at 49 °C for 15 min) renders the TFIID inactive [6, 8]. We demonstrate that: 1) Immunodepletion of nuclear extracts with an anti-TFII-I antibody completely abrogates transcription of the TATAϪInrϩ V promoter, which is restored by addition of purified TFII-I. These antibodies have no effect on the TATAϩInrϪ IgH promoter. Transcriptional complementation assays using heat-treated nuclear extracts demonstrate that TFIID is both necessary and sufficient for a TATAϩInrϪ promoter, TFII-I is required for the TATAϪInrϩ V promoter
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