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Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

ABSTRACT: Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

SUBMITTER: Nguyen VQ

PROVIDER: S-EPMC8420877 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

Nguyen Vu Q VQ Ranjan Anand A Liu Sheng S Tang Xiaona X Ling Yick Hin YH Wisniewski Jan J Mizuguchi Gaku G Li Kai Yu KY Jou Vivian V Zheng Qinsi Q Lavis Luke D LD Lionnet Timothée T Wu Carl C

Molecular cell 20210809 17

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On ...[more]

PMID: 34375585

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Project description:UnlabelledDuring their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta- and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta- and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses.ImportanceControl of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC).

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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Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

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Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

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