The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions

RNA polymerases (RNAPs) require basal transcription factors to assist them during transcription initiation. One of these factors, TFIIB, combines promoter recognition, recruitment of RNAP, promoter melting, start site selection and various post-initiation functions. The ability of 381 site-directed mutants in the TFIIB ‘linker domain’ to stimulate abortive transcription was systematically quantitated using promoter-independent dinucleotide extension assays. The results revealed two distinct clusters (mjTFIIB E78-R80 and mjTFIIB R90-G94, respectively) that were particularly sensitive to substitutions. In contrast, a short sequence (mjTFIIB A81-K89) between these two clusters tolerated radical single amino acid substitutions; short deletions in that region even caused a marked increase in the ability of TFIIB to stimulate abortive transcription (‘superstimulation’). The superstimulating activity did, however, not correlate with increased recruitment of the TFIIB/RNAP complex because substitutions in a particular residue (mjTFIIB K87) increased recruitment by more than 5-fold without affecting the rate of abortive transcript stimulation. Our work demonstrates that highly localized changes within the TFIIB linker have profound, yet surprisingly disconnected, effects on RNAP recruitment, TFIIB/RNAP complex stability and the rate of transcription initiation. The identification of superstimulating TFIIB variants reveals the existence of a previously unknown rate-limiting step acting on the earliest stages of gene expression

Molecular mechanisms of transcription initiation—structure, function, and evolution of TFE/TFIIE-like factors and open complex formation

Transcription initiation requires that the promoter DNA is melted and the template strand is loaded into the active site of the RNA polymerase (RNAP), forming the open complex (OC). The archaeal initiation factor TFE and its eukaryotic counterpart TFIIE facilitate this process. Recent structural and biophysical studies have revealed the position of TFE/TFIIE within the pre-initiation complex (PIC) and illuminated its role in OC formation. TFE operates via allosteric and direct mechanisms. Firstly, it interacts with the RNAP and induces the opening of the flexible RNAP clamp domain, concomitant with DNA melting and template loading. Secondly, TFE binds physically to single-stranded DNA in the transcription bubble of the OC and increases its stability. The identification of the β-subunit of archaeal TFE enabled us to reconstruct the evolutionary history of TFE/TFIIE-like factors, which is characterised by winged helix (WH) domain expansion in eukaryotes and loss of metal centres including iron-sulfur clusters and Zinc ribbons. OC formation is an important target for the regulation of transcription in all domains of life. We propose that TFE and the bacterial general transcription factor CarD, although structurally and evolutionary unrelated, show interesting parallels in their mechanism to enhance OC formation. We argue that OC formation is used as a way to regulate transcription in all domains of life, and these regulatory mechanisms coevolved with the basal transcription machinery

High-resolution mutagenesis of the linker domain of archaeal basal transcription factor TFIIB

TFIIB is a component of the minimal eukaryotic as well as the archaeal transcriptional machinery that is essential for promoter-directed transcription. The flexible linker domain of the protein engages in an intimate association with the RNA polymerase surface. Early structural data suggested that the linker forms a finger-like structure (the ‘B-finger’) within the RNA exit channel projecting into the active centre. Biochemical data indicated a contribution of the archaeal ‘B-finger’ domain to the catalytic mechanism by stimulating abortive transcription. The path of the linker within the RNA polymerase catalytic centre has recently been re-assessed and residues of the original Bfinger were re-assigned to structural elements named ‘B-reader helix’ and ‘B-reader loop’. Novel high-throughput tools, in combination with a comprehensive mutagenesis screen of residues E78 to A95, facilitated the biochemical evaluation of structural-functional relationships of the M. jannaschii TFIIB linker – RNAP interface at a single residue resolution. The performance of such point mutants during abortive initiation and RNA polymerase recruitment was interpreted in light of structural information. The tip region of the ‘B-finger’ that was predicted to be closest to the active site was insensitive to mutations in abortive initiation assays, thus disproving the original model. Individual residues, forming part of the B-reader helix and the C-terminal half of the Breader loop, were found to engage in abortive transcription. Three-residue deletions within the N-terminal half of the B-reader loop resulted in super-stimulation of abortive transcription. Individual point mutations within the B-reader loop led to enhanced recruitment of RNA polymerase. A functional role of the loop in stabilizing TFIIBRNA polymerase-DNA complexes in both the absence and presence of TBP seems feasible. The combined data provide a detailed view of biochemical functions of individual residues of the TFIIB linker favouring the ‘B-reader’ model over the ‘B-finger’ model

Two Modes of Transcriptional Activation at Native Promoters by NF-κB p65

The NF-κB family of transcription factors is crucial for the expression of multiple genes involved in cell survival, proliferation, differentiation, and inflammation. The molecular basis by which NF-κB activates endogenous promoters is largely unknown, but it seems likely that it should include the means to tailor transcriptional output to match the wide functional range of its target genes. To dissect NF-κB–driven transcription at native promoters, we disrupted the interaction between NF-κB p65 and the Mediator complex. We found that expression of many endogenous NF-κB target genes depends on direct contact between p65 and Mediator, and that this occurs through the Trap-80 subunit and the TA1 and TA2 regions of p65. Unexpectedly, however, a subset of p65-dependent genes are transcribed normally even when the interaction of p65 with Mediator is abolished. Moreover, a mutant form of p65 lacking all transcription activation domains previously identified in vitro can still activate such promoters in vivo. We found that without p65, native NF-κB target promoters cannot be bound by secondary transcription factors. Artificial recruitment of a secondary transcription factor was able to restore transcription of an otherwise NF-κB–dependent target gene in the absence of p65, showing that the control of promoter occupancy constitutes a second, independent mode of transcriptional activation by p65. This mode enables a subset of promoters to utilize a wide choice of transcription factors, with the potential to regulate their expression accordingly, whilst remaining dependent for their activation on NF-κB

The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation

TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP “clamp” region as a regulatory hot spot for both transcription initiation and transcription elongation

Regulation of transcript elongation through cooperative and ordered recruitment of cofactors

We studied the regulation of murine CD80, a gene whose basal transcriptional status was characterized by the presence of a stalled RNA polymerase II complex on the promoter-proximal region. Stimulus-induced activation of productive elongation involved a complex interplay of regulated events that included a synergy between ordered cofactor recruitment. This cascade of recruitments was initiated through the engagement of transcription factor NF-κB, leading to the temporal association of histone acetyltransferases and the consequent selective acetylation of a transcription start site downstream nucleosome. This in turn culminated into the nucleosomal association of Brd4-associated P-TEFb, a protein complex containing kinase specific for serine 2 of Rbp 1, the largest subunit of the carboxyl-terminal domain of RNA polymerase II. The consequent phosphorylation of serine 2 residues in CTD by CDK9 in the P-TEFb complex then facilitated escape of polymerase II into the productive elongation phase. Thus, the cooperative mechanisms that integrate between independent pathways characterize regulation of the elongation step of transcription, thereby providing another level at which specificity of gene regulation can be achieved

Elucidation of Mechanisms Underlying Metastatic Melanoma Immune Escape via Suppression of Major Histocompatibility Complex (MHC) II through Dysregulation of the JAK/STAT Pathway

Transcriptional activation of Major Histocompatability Complex (MHC) I and II molecules by the cytokine interferon gamma (IFN-g) is a key step in cell-mediated immunity against pathogens and tumors. Following IFN-g induction, JAK/STAT signaling triggers activation of MHC genes. Recent evidence suggests suppression of MHC I and II expression on multiple tumor types plays important roles in tumor immunoevasion. One such tumor is malignant melanoma, the leading cause of skin cancer related deaths. Despite awareness of MHC expression defects, the molecular mechanisms by which melanoma cells suppress MHC and escape from immune-mediated destruction remain unknown. Here we analyze dysregulation of the JAK/STAT pathway and its role in suppression of MHC II in melanoma cell lines at the Radial Growth Phase (RGP), the Vertical Growth Phase (VGP) and the Metastatic Phase (MET). RGP and VGP cells express both MHC II and the MHC master regulator, the Class II Transactivator (CIITA). MET cells lack not only MHC II and CIITA, but also both STAT 1 and the STAT 1 coactivator, the Interferon Response Factor (IRF) 1. Our studies have implicated that the suppression of MHCII on the cell surface of metastatic melanoma is due to silencing at the level of STAT1 transcription. Furthermore, we determined that silencing of STAT1 is, in part, due to hemi-methylation of the STAT1 promoter

The Effects of the histones and histone-interacting partners on transcriptional regulation

Ph.DDOCTOR OF PHILOSOPH

Functional interactions of the Transcription Factor B during transcription initiation in Pyrococcus furiosus

The preinitiation complex of the transcription machinery in archaeal organisms resembles a simplified version of the eukaryotic RNA polymerase II transcription system. Both systems share homologous general transcription factors to recruit RNA polymerase to the promoter to initiate RNA synthesis. The transcription factor (II)B plays an important role during transcription initiation. Based on eukaryotic cryo-EM and crystal structures several functional interactions and structural transitions of TF(II)B were proposed. To detect specific interactions of the archaeal P. furiosus TFB during transcription initiation different in vitro transcription assays were performed. In addition, the replication protein A of P. furiosus was also investigated using various in vitro experiments. Crosslinking experiments using TFB, which contained a UV inducible photo crosslinker, and site-specific radioactively labeled DNA templates revealed an almost similar topology of the archaeal TFB B-reader and B-linker domains in the preinitiation complex in comparison to corresponding regions predicted in eukaryotic structures. Unlike it was postulated in open complex models, the non-transcribed strand is located closer to the B-linker strand than the B-linker helix. The B-core amino acid F192 contact DNA 19 nucleotides upstream the transcribed strand, in accordance to a published crystal of P. woesei TATA/TBP/TFB-core structure, but is different to predicted eukaryotic closed and open complex models. Crosslinking experiments in stalled complexes showed that RNA interacts with the B-reader loop at a length of 6nt, and further clashes with the B-reader helix domain with a length of 8nt. At register +10 the TFB B-reader is displaced, which causes collapse of the transcription bubble. It was also demonstrated that TFB is present at register +6 to +14 in the complex, and tended to be released from register +15 onwards, indicating a destabilization of TFB at register +13/+14. Alanine substitutions of amino acids of the TFB B-reader loop revealed that this region mainly stabilizes the transcription bubble due to charge-dependent interactions with the transcribing strand. In contrast to the predicted RNA-DNA separation model derived from a eukaryotic initially transcribing complex, RNA-strand separation does not depend on the charge of the PfuTFB B-reader loop. Single molecule FRET experiments revealed that DNA bending depends on the presence of TFB in P. furiosus. In vitro transcription assays with RPA showed that this protein has binding preference to single stranded DNA. Experiments further showed that RPA is not involved in transcription initiation, but it stimulates transcription. Therefore RPA functions during elongation of transcription, possibly due to a stabilization of the RNA polymerase and increase of the processivity. The results presented here give a more detailed insight into molecular interactions of TFB and are the first biochemical data on dynamic rearrangements of TFB during transcription initiation and transition to early elongation. It further deepens the understanding of archaeal transcription processes and complements structural information derived from related eukaryotic organisms