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Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB.

ABSTRACT: The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2 binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2 binding reduces backbone mobility in the b-turn (Arg28-Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2-dependent structural changes. These structural effects of Zn2 binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II.

SUBMITTER: Ghosh M

PROVIDER: S-EPMC1223989 | biostudies-other | 2004 Mar

REPOSITORIES: biostudies-other

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Project description:The action of oestrogen hormones is mediated through the oestrogen receptor (ER), a member of a large superfamily of nuclear receptors that function as ligand-activated transcription factors. Sequence-specific transcription factors, including the nuclear receptor superfamily, are thought to interact either directly or indirectly with general transcription factors to regulate transcription. Although numerous studies have focused on the identification of potential co-activators interacting with isolated trans-activation domains of ER, few have investigated the mechanisms by which ER transmits its signal to the basal transcription machinery. We show that ER does not stabilize the binding of the TATA-box binding protein (TBP) of the TFIID complex, or of TFIIB to the promoter, although a stable ER-TBP-TFIIB-promoter complex was detected, suggesting that ER, TBP and TFIIB might interact with each other to form a complex to the promoter. We also demonstrate that ER binds specifically to TFIIB, a key component of the preinitiation complex. Affinity chromatography with immobilized deletion mutants of ER maps a TFIIB interaction region that encompasses the DNA-binding domain. The addition of excess TFIIB to transcription reactions in vitro did not, however, affect the magnitude of transcriptional activation by ER. These results indicate that, in contrast with current models, ER does not activate transcription by increasing the rate of assembly of TFIIB into the transcription complex. An increased concentration of TFIIB was unable, by itself, to overcome the requirement for ER. By using an immobilized promoter-template assay employing nuclear extract from HeLa cells, recombinant human ER increased the stable association of subsequent components of the transcription machinery (TFIIE and TFIIF), in correlation with ER-induced transcription. Our results suggest that ER acts, in an early step, during or immediately after the formation of template-committed complexes containing TFIIB, favouring the recruitment of one or more components of the basic transcription machinery as well as co-activators.

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Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB.

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