Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

156

160

The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors. The mammalian target of rapamycin (mTOR)3 is a central metabolic sensor that coordinates cell growth and proliferation with the availability of growth factors, nutrients, and energy sufficiency. mTOR exists in two multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (1-3). Under conditions of rapid growth and proliferation, mTOR stimulates several anabolic processes, including mRNA translation, transcription, and lipid biosynthesis. The mTORC1 is well known to enhance cap-dependent translation initiation through the direct phosphorylation of S6K1 and 4E-BP1 in response to mitogen and nutrient stimulation (4). The more recently discovered mTORC2 can directly phosphorylate AKT and conventional protein kinase C and is involved in maintenance of actin cytoskeleton in a yet to be defined mechanism. mTOR is a major signaling component of the phosphatidylinositol 3-kinase/AKT pathway that is most frequently dysregulated in cancer (3, 5-7).In addition to the direct control of translational apparatus by mTORC1, several reports have implicated the mTOR signaling in the molecular events occurring the nucleus, such as RNA polymerase (Pol) I transcription, essential for the ribosomal biogenesis and accumulation of cell mass (8 -10). The genetic and biochemical studies in bu...

Section: Maf1 Depletion Increases Basal Trna Transcription and Rendersupporting

confidence: 90%

Requirement of the mTOR Kinase for the Regulation of Maf1 Phosphorylation and Control of RNA Polymerase III-dependent Transcription in Cancer Cells

Shor

Wu²,

Shakey³

et al. 2010

156

160

“…4A (left panel), weak binding of Brf2-TFIIIB to U6 pro- moter probes is observed when all three components are included in the DNA binding reaction, whereas no binding was observed in any reactions that did not contain TBP, consistent with the observation that Brf2-TFIIIB is TATA-box dependent (data not shown). Weak, but nonetheless cooperative, DNA binding is also observed for TBP plus Brf2 at levels greater than that observed for either factor alone, as previously described (20,31,32). In contrast, SNAP C ␥4 bound well and effectively recruited TBP to DNA (Fig.…”

Section: The Cooh-terminal Region Of Snap50 Is Required For Dnasupporting

confidence: 81%

The Unorthodox SNAP50 Zinc Finger Domain Contributes to Cooperative Promoter Recognition by Human SNAPC

Jawdekar

Hanzlowsky

Hovde

et al. 2006

C through its zinc finger domain. The SNAP50 zinc finger domain contains 15 cysteine and histidine residues configured in two potential zinc coordination arrangements. Individual alanine substitution of each cysteine and histidine residue demonstrated that eight sites are important for DNA binding by SNAP C . However, metal binding studies revealed that SNAP C contains a single zinc atom indicating that only one coordination site functions as a zinc finger. Of the eight residues critical for DNA binding, four cysteine residues were also essential for both U1 and U6 transcription by RNA polymerase II and III, respectively. Surprisingly, the remaining four residues, although critical for U1 transcription could support partial U6 transcription. DNA binding studies showed that defects in DNA binding by SNAP C alone could be suppressed through cooperative DNA binding with another member of the RNA polymerase III general transcription machinery, TFIIIB. These results suggest that these eight cysteine and histidine residues perform different functions during DNA binding with those residues involved in zinc coordination likely performing a dominant role in domain stabilization and the others involved in DNA binding. These data further define the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding domain.The human snRNA 3 gene family is unusual in that related member genes are transcribed by either RNA polymerase II or III depending upon their core promoter structures, and they thus serve as an interesting model to understand principles of polymerase selection and activity both during normal and deregulated growth (reviewed in Refs. 1 and 2). Human snRNA genes are defined by the presence of a diagnostic promoter element called the proximal sequence element (PSE). For both polymerase systems, the PSE recruits the general transcription factor called SNAP C (3), which is also known as PTF (4).SNAP C is composed of at least five subunits SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19 (5-9). SNAP190 can interact with all the other subunits, and provides a central architectural backbone to the complex (10). SNAP190 directly interacts with the transcriptional activator protein Oct-1 during stimulated transcription of human snRNA genes (11, 12). In the RNA polymerase III pathway, SNAP190 also interacts with TBP to recruit Brf2-TFIIIB to the TATA box of human U6 snRNA genes (13,14). Thus, the upstream signal from Oct-1 is conveyed through SNAP190 stimulating preinitiation complex assembly with other general transcription factors as a prerequisite for RNA polymerase III recruitment. Interestingly, protein kinase CK2 can phosphorylate SNAP190 to impede DNA binding; however, promoter recognition by SNAP C can be restored by cooperative binding of TBP to those promoter sequences containing both a PSE and TATA box, but not sequences lacking the TATA element (15). This last observation suggests that CK2 can differentially influence RNA polymerase II and III snRNA transcription by covalent modification of SNAP C...

“…S2). If this Bdp1-Brf1 interface is retained in vertebrate Brf1, it is noteworthy that this part of the Brf1 helix H24 is clearly absent from the Brf1 paralogue Brf2 (34). A prior in vivo analysis (35) of the effect of sequence substitutions in 19 residues of Brf1 between amino acid residues 453 and 508 did not reveal any growth defect that could be ascribed to a defect in interaction with Bdp1.…”

Section: Discussionmentioning

confidence: 99%

Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Kassavetis

Driscoll²,

Geiduschek

2006

The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIBrelated half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding. An extensive collection of N-terminal, C-terminal, and internal deletion proteins has been used to demarcate the interacting Bdp1 domain to a 66-amino acid segment that includes the SANT domain of this subunit and is phylogenetically the most conserved region of Bdp1.The RNA polymerase (pol) 3 III transcription apparatus of yeast comprises, in addition to the polymerase itself, three transcription initiation factors, TFIIIA, -B, and -C.The three-subunit TFIIIB is the core initiation factor required and sufficient for recruiting pol III to the promoter and accurately initiating transcription. The large six-subunit TFIIIC places TFIIIB on DNA upstream of the transcriptional start site and protects the generally very small pol III transcription units from encroachment by repressive chromatin (1, 2). TFIIIA is the 5 S rRNA gene-specific factor that places TFIIIC on the transcription unit-internal promoter of these genes (reviewed in Refs. 3-5).The work that is presented here deals with the structure of TFIIIB, which is composed of three subunits: the TATA-binding protein (TBP), Brf1, and Bdp1. TBP and Brf1 bind tightly and co-purify, whereas Bdp1 dissociates during later steps of conventional column chromatography. (The two separated fractions of TFIIIB have been designated BЈ and BЉ, and B double prime has been adopted as the eponym of the Bdp1 subunit.) The 596-amino acid Brf1 is a fusion protein, with an N-terminal half that is related to the pol II transcription initiation factor TFIIB and a pol III-specific C-terminal half. A conserved segment of the C-terminal half (homology domain II; amino acids 439 -515) constitutes the Brf1 high affinity binding site for the evolutionarily highly conserved core of TBP (comprising all but the N-terminal 60 amino acids of the Saccharomyces cerevisiae protein); the N-terminal half of Brf1 engages TBP through a separate lower affinity interaction.The N-and C-terminal halves of Brf1 also provide separate Bdp1-docking sites, with the C-proximal homology domain II contributing the higher Bdp1 affinity. TBP makes only a minor direct contribution to bringing Bdp1 into TFIIIB. Thus, the pol III-specific half of Brf1 appears to contribute the interactions that principally hold TFIIIB together. Ind...