Dataset Information

Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II.

ABSTRACT: The general transcription factor TFIIB has a central role in the assembly of the preinitiation complex at the promoter, providing a platform for the entry of RNA polymerase II/TFIIF. We used an RNA interference (RNAi)-based system in which TFIIB expression is ablated in vivo and replaced with a TFIIB derivative that contains a silent mutation and is refractory to the RNAi. Using this approach, we found that transcriptionally defective TFIIB amino-terminal mutants showed distinct effects on the basis of their ability to compete with wild-type TFIIB in vivo. Moreover, analysis of the TFIIB mutant derivatives by chromatin immunoprecipitation showed that promoter occupancy by TFIIB is dependent on the association with RNA polymerase II. Together, our results support a mode of preinitiation complex assembly in which TFIIB/RNA polymerase II recruitment to the promoter occurs in vivo.

SUBMITTER: Elsby LM

PROVIDER: S-EPMC1559668 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II.

Elsby Laura M LM O'Donnell Amanda J M AJ Green Laura M LM Sharrocks Andrew D AD Roberts Stefan G E SG

EMBO reports 20060728 9

The general transcription factor TFIIB has a central role in the assembly of the preinitiation complex at the promoter, providing a platform for the entry of RNA polymerase II/TFIIF. We used an RNA interference (RNAi)-based system in which TFIIB expression is ablated in vivo and replaced with a TFIIB derivative that contains a silent mutation and is refractory to the RNAi. Using this approach, we found that transcriptionally defective TFIIB amino-terminal mutants showed distinct effects on the b ...[more]

PMID: 16878124

Similar Datasets

Project description:Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

Dataset Information

Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II.

Publications

Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets