TFIIE Research Articles

GTF2E2 downregulated by miR-340-5p inhibits the malignant progression of glioblastoma.

Glioblastoma is the most common malignant tumor in the central nervous system. The general transcription factor IIE subunit beta (GTF2E2) is crucial for physiological and pathological functions, but its roles in the malignant biological function of glioma remain ambiguous. CCK-8, colony formation assays, TUNEL assays, cell migration assays, wound-healing assays, and xenograft model were established to investigate the biological functions of GTF2E2 both in vitro and in vivo. GTF2E2 was overexpressed in glioma and was associated with poor prognosis of glioma patients. Biological functions of GTF2E2 were investigated both in vitro and in vi0vo by multiple experiments. Moreover, we explored the possible mechanisms of GTF2E2. In our results, we demonstrated that GTF2E2 could be regulated by miR-340-5p directly or indirectly. CCND1 was transcriptionally affected by GTF2E2 and glioma progression was then regulated. Our data presented the overexpression of GTF2E2 in glioma and indicated the association between GTF2E2 and glioma prognosis. GTF2E2 was found to be regulated by miR-340-5p and thus affect downstream gene expressions and glioma progression. Our results indicate that GTF2E2 might be a potential target in the diagnosis and treatments of glioblastoma.

GTF2E2 is a novel biomarker for recurrence after surgery and promotes progression of esophageal squamous cell carcinoma via miR-139-5p/GTF2E2/FUS axis

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal gastrointestinal malignancies with high mortality. Recurrence develops within only a few years after curative resection and perioperative adjuvant therapy in 30–50% of these patients. Therefore, it is essential to identify postoperative recurrence biomarkers to facilitate selecting the following surveillance and therapeutic strategies. The general transcription factor IIE subunit beta (GTF2E2) is crucial for physiological and pathological functions, but its roles in the aggression and recurrence of ESCC remain ambiguous. In this study, we found that GTF2E2 was highly expressed in ESCC samples, and elevated GTF2E2 expression predicted early recurrence after surgery for ESCC patients. High expression of GTF2E2 associated with more aggressive clinic features and poor prognosis. GTF2E2 promoted the proliferation and mobility of ESCC cells in vitro and in vivo. We further revealed that miR-139-5p repressed GTF2E2 expression by downregulating its mRNA through binding with Argonaute 2 (Ago2). Rescue assays suggested that miR-139-5p affected GTF2E2-mediated ESCC progression. Moreover, GTF2E2 positively interacted with FUS promoter and regulated FUS expression, and the phenotype changes caused by GTF2E2 manipulation were recovered by rescuing FUS expression in ESCC cells. Additionally, we demonstrated that GTF2E2 promotes ESCC cells progression via activation of the AKT/ERK/mTOR pathway. In conclusion, GTF2E2 may serve as a novel biomarker for recurrence after surgery and a potential therapeutic target for ESCC patients, and it promotes ESCC progression via miR-139-5p/GTF2E2/FUS axis.

MicroRNA 452 regulates GTF2E1 expression in colorectal cancer cells

MicroRNAs play important roles in the pathogenesis of human diseases by regulating target gene expression in specific cells or tissues. Previously, we identified microRNA 452 (MIR452), which was specifically upregulated in early stage of human colorectal cancer (CRC) tissues. Here, we show the biological role of MIR452 and general transcription factor IIE subunit 1 (GTF2E1) in colorectal cancer. A luciferase reporter system was used to confirm the effect of MIR452 on GTF2E1 expression. The expression levels of MIR452 and the target genes were evaluated by quantitative RT-PCR (qRT-PCR) and western blotting. We verified the association between MIR452 and the GTF2E1 expression and found that GTF2E1 transcripts were directly downregulated by MIR452. The mRNA and protein levels of GTF2E1 were also downregulated in CRC cells upon transfection with MIR452. GTF2E1 protein expression was decreased in CRC tissues compared to adjacent nontumour tissues. These results suggest that MIR452 might directly or indirectly regulate the genes transcription related to CRC by downregulating GTF2E1 expression.

Dynamics of the Extended String-Like Interaction of TFIIE with the p62 Subunit of TFIIH

General transcription factor II E (TFIIE) contains an acid-rich region (residues 378–393) in its α-subunit, comprising 13 acidic and two hydrophobic (Phe387 and Val390) residues. Upon binding to the p62 subunit of TFIIH, the acidic region adopts an extended string-like structure on the basic groove of the pleckstrin homology domain (PHD) of p62, and inserts Phe387 and Val390 into two shallow pockets in the groove. Here, we have examined the dynamics of this interaction by NMR and molecular dynamics (MD) simulations. Although alanine substitution of Phe387 and/or Val390 greatly reduced binding to PHD, the binding mode of the mutants was similar to that of the wild-type, as judged by the chemical-shift changes of the PHD. NMR relaxation dispersion profiles of the interaction exhibited large amplitudes for residues in the C-terminal half-string in the acidic region (Phe387, Glu388, Val390, Ala391, and Asp392), indicating a two-site binding mode: one corresponding to the final complex structure, and one to an off-pathway minor complex. To probe the off-pathway complex structure, an atomically detailed free-energy landscape of the binding mode was computed by all-atom multicanonical MD. The most thermodynamically stable cluster corresponded to the final complex structure. One of the next stable clusters was the off-pathway structure cluster, showing the reversed orientation of the C-terminal half-string on the PHD groove, as compared with the final structure. MD calculations elucidated that the C-terminal half-acidic-string forms encounter complexes mainly around the positive groove region with nearly two different orientations of the string, parallel and antiparallel to the final structure. Interestingly, the most encountered complexes exhibit a parallel-like orientation, suggesting that the string has a tendency to bind around the groove in the proper orientation with the aid of Phe387 and/or Val390 to proceed smoothly to the final complex structure.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

P53 and TFIIEα share a common binding site on the Tfb1/p62 subunit of TFIIH

The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the alpha-subunit of the general transcription factor IIE (TFIIEalpha). We have determined that the acidic carboxyl terminus of TFIIEalpha (TFIIEalpha(336-439)) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEalpha binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEalpha(336-439) is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEalpha(336-439) and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEalpha(336-439) contains a small domain (residues 395-433) folded in a novel betabetaalphaalphaalpha topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for TFIIEalpha(336-439) binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEalpha(336-439) can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEalpha and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53.

Investigation of molecular size of transcription factor TFIIE in solution

Human general transcription factor IIE (TFIIE), a component of a transcription preinitiation complex associated with RNA polymerase II, was characterized by size-exclusion chromatography, mass spectrometry, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS). Recombinant human TFIIE was purified to homogeneity and shown to contain equimolar amounts of TFIIEalpha (50 kDa) and TFIIEbeta (35 kDa) by SDS-PAGE. In the analysis of size-exclusion chromatography of the purified sample, as already reported, TFIIE was shown to be a 170-kDa alpha(2)beta(2) heterotetramer. However, by using electrospray ionization mass spectrometry the purified sample gave the molecular mass of 84,152 +/- 5, indicating that TFIIE is an alphabeta heterodimer but not a heterotetramer. Analytical ultracentrifugation experiment of TFIIE provided that only a single component with the molecular mass of ca. 80,000 existed in solution, also suggesting an alphabeta heterodimer. In addition, its extraordinarily rod-like molecular shape was confirmed by SAXS. It is likely that the rod-like molecular shape of TFIIE has misled larger molecular size in size-exclusion chromatography, which was calibrated by globular proteins. It is demonstrated that TFIIE exists as a heterodimer under our present conditions in solution, although two molecules of heterodimer might be required for the formation of the preinitiation complex with RNA polymerase II for starting the transcription process.

Efficient production of recombinant human transcription factor IIE

Transcription factor IIE (TFIIE) is a general initiation and promoter escape factor for RNA polymerase II composed of p56 (TFIIE-α) and p34 (TFIIE-β) subunits. Our laboratories experienced difficulty producing adequate quantities of recombinant human TFIIE-α for in vitro studies using available clones. We therefore re-engineered the TFIIE subunit production vectors and tested various Escherichia coli host strains to optimize expression. We report a much-improved system for production of pure, soluble, and active TFIIE complex for in vitro studies.

Mediator Influences Schizosaccharomyces pombe RNA Polymerase II-dependent Transcription in Vitro

The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.

Artificial recruitment of certain Mediator components affects requirement of basal transcription factor IIE.

Basal transcription factors are essential for RNA polymerase II (RNAPII)-catalysed transcription of many but not all the mRNA-encoding genes in vivo as well as in vitro. For example, copper-inducible transcription of the copper metallothionein gene CUP1 occurs independently of basal factor TFIIE in budding yeast. To gain insight into the mechanism by which the requirement for TFIIE is bypassed, we artificially recruited certain constituents of Mediator, a large protein complex transmitting signals from various activators to the RNAPII machinery, to the CUP1 promoter by protein fusions with Ace1, the copper-inducible activator. Fusions with Med2 or Pgd1 activated CUP1 independently of TFIIE. Surprisingly, fusions with neither Srb5 nor Med9 circumvented TFIIE requirement for the CUP1 activation. Components of TFIID were similarly recruited to the CUP1 promoter without activation. By using a chromatin immunoprecipitation technique, we found that TFIIE is necessary for stable binding of TFIIH and RNAPII to the ADH1 promoter, whose activation requires TFIIE. However, binding of TFIIH and RNAPII to CUP1 upon its activation did not require TFIIE. Our results strongly suggest that the TFIIE requirement of a gene is determined by a target(s) in Mediator through which the signal of the cognate activator is transmitted.

Functional Connections between Mediator Components and General Transcription Factors of Saccharomyces cerevisiae

The yeast Gal11 protein is an important component of the Mediator complex in RNA polymerase II-directed transcription. Gal11 and the general transcription factor (TF) IIE are involved in regulation of the protein kinase activity of TFIIH that phosphorylates the carboxyl-terminal domain of RNA polymerase II. We have previously shown that Gal11 binds the small and large subunits of TFIIE at two Gal11 domains, A and B, respectively, which are important for normal function of Gal11 in vivo. Here we demonstrate that Gal11 binds directly to TFIIH through domain A in vitro. A null mutation in GAL11 caused lethality of cells when combined with temperature-sensitive mutations in the genes encoding TFIIE or the carboxyl-terminal domain kinase, indicating the presence of genetic interactions between Gal11 and these proteins. Mutational depletion of Gal11 or TFIIE caused inefficient opening of the transcription initiation region, but had no significant effect on TATA-binding protein occupancy of the TATA sequence in vivo. These results suggest that the functions of Gal11 and TFIIE are necessary after recruitment of TATA-binding protein to the TATA box presumably at the step of stable preinitiation complex formation and/or promoter melting. We illustrate genetic interactions between Gal11 and other Mediator components such as Med2 and Pgd1/Hrs1/Med3.

Transcription-coupled DNA repair in yeast transcription factor IIE (TFIIE) mutants

We examined the role of yeast transcription initiation factor IIE (TFIIE) in eukaryotic transcription-coupled repair (TCR), the preferential removal of DNA damage from the transcribed strands of genes over non-transcribed sequences. TFIIE can recruit the transcription initiation/repair factor TFIIH to the RNA polymerase II (RNA pol II) initiation complex to facilitate promoter clearance. Following exposure to UV radiation, the RNA pol II elongation complex is blocked at sites of UV-induced DNA damage, and may be recognized by nucleotide excision repair proteins, thus enabling TCR. The TFA1 gene encodes the large subunit of TFIIE. We determined how DNA repair is affected by TFA1 conditional mutations. In particular, we find proficient TCR in a heat-sensitive tfa1 mutant at the non-permissive temperature during which growth is inhibited and overall RNA pol II transcription is reported to be inhibited. We demonstrate that transcription of the RPB2 gene was reduced, but readily detectable, in the heat-sensitive tfa1 mutant at the non-permissive temperature and thereby prove that TCR does occur in an expressed gene in the absence of TFIIE in vivo. We demonstrate that TCR occurs even at low levels of transcription.

Activator-Specific Requirement for the General Transcription Factor IIE in Yeast

The general transcription factor (TF) IIE is required for mRNA synthesis of many, but not all, genes in yeast. In the transcription process, TFIIE regulates TFIIH kinase activity that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD and the CTD kinase Kin28, a subunit of TFIIH, have been shown to be dispensable for activation of several heat shock genes and the copper metallothionein gene CUP1. Here we analyzed requirement of TFIIE for transcription of these genes and found that TFIIE is necessary for activation of the heat shock genes by heat shock transcription factor Hsf1. By contrast, transcription of CUP1 mediated by both Hsf1 and copper-activated transcription factor Ace1 was inducible after inactivating TFIIE. These results show that both TFIIE and the CTD/the CTD kinase exhibit “gene specificities” which are overlapping, but not identical to each other, and thereby suggest that TFIIE functions with or without involvement of the CTD/the CTD kinase depending on the gene to be transcribed.

The general transcription factors IIA, IIB, IIF, and IIE are required for RNA polymerase II transcription from the human U1 small nuclear RNA promoter.

RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription.

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Functional Correlation among Gal11, Transcription Factor (TF) IIE, and TFIIH in Saccharomyces cerevisiae: Gal11 AND TFIIE COOPERATIVELY ENHANCE TFIIH-MEDIATED PHOSPHORYLATION OF RNA POLYMERASE II CARBOXYL-TERMINAL DOMAIN SEQUENCES

Saccharomyces cerevisiae Gal11, a component of the holoenzyme of RNA polymerase II, interacts through its functional domains A and B with the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE, respectively. We have recently suggested that Gal11 functions through a common pathway with TFIIE in transcriptional regulation (Sakurai, H., and Fukasawa, T. (1997) J. Biol. Chem. 272, 32663-32669). Here, we report that the activity of the TFIIH-associated kinase, responsible for phosphorylation of the largest subunit of RNA polymerase II at the carboxyl-terminal domain (CTD), is enhanced cooperatively by Gal11 and TFIIE. The enhancement of CTD phosphorylation was observed in the holoenzyme of RNA polymerase II, but not in its core enzyme. The stimulatory effect was completely abolished in the absence of either domain B of Gal11 or the Tfa1 subunit of TFIIE, suggesting that the domain B-Tfa1 interaction is necessary, if not sufficient, for an extensive phosphorylation of the CTD by TFIIH. Stimulation of basal transcription by Gal11 was coupled with enhancement of TFIIH-catalyzed CTD phosphorylation in a cell-free transcription system, suggesting that Gal11 activates transcription by stimulating the CTD phosphorylation in the cell.

A Debilitating Mutation in Transcription Factor IIE with Differential Effects on Gene Expression in Yeast

The influence of transcription factor (TF) IIE on mRNA synthesis in vivo was examined in a temperature-sensitive yeast mutant. A missense mutation in the conserved zinc finger domain severely weakened TFIIE's transcription activity without appreciably affecting its quaternary structure, chromatographic properties, or cellular abundance. The mutation conferred recessive slow-growth and heat-sensitive phenotypes in yeast, but quantitative effects on promoter utilization by RNA polymerase II ranged from strongly negative to somewhat positive. Heat-induced activation of the HSP26, HSP104, and SSA4 genes was attenuated in the mutant, indicating dependence on TFIIE for maximal rates of de novo synthesis. Constitutive HSP expression in mutant cells was elevated, exposing a negative (likely indirect) influence by TFIIE in the absence of heat stress. Our results corroborate and extend recent findings of differential dependence on TFIIE activity for yeast promoters, but reveal an important counterpoint to the notion that dependence is tied to TATA element structure (Sakurai, H., Ohishi, T., and Fukasawa, T. (1997) J. Biol. Chem. 272, 15936-15942). We also provide empirical evidence for conservation of structure-activity relationships in TFIIE's zinc finger domain, and establish a direct link between TFIIE's biochemical activity in reconstituted transcription and its function in cellular mRNA synthesis.

Yeast Gal11 and Transcription Factor IIE Function through a Common Pathway in Transcriptional Regulation

The global transcription regulator Gal11, a component of RNA polymerase II holoenzyme, is required for full expression of many genes in yeast. We previously reported that Gal11 binds the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE through Gal11 functional domains A and B, respectively. Here we demonstrate that the C-terminal basic region in Tfa2 is responsible for binding to domain A, whereas both the N-terminal hydrophobic and internal glutamic acid-rich regions in Tfa1 are responsible for binding to domain B. Yeast cells bearing a C-terminal deletion encompassing the Gal11-interacting region in each of the two TFIIE subunits, being viable, exhibited no obvious phenotype. In contrast, combination of the two deletions (TFIIE-DeltaC) showed phenotypes similar to those of gal11 null mutations. The levels of mRNA from TATA-containing genes, but not from TATA-less genes, decreased in TFIIE-DeltaC to an extent comparable to that in the gal11 null mutant. Combination of TFIIE-DeltaC with a gal11 null mutation did not result in an enhanced effect, suggesting that both TFIIE and Gal11 act in a common regulatory pathway. In a reconstituted cell-free system, Gal11 protein stimulated basal transcription in the presence of wild-type TFIIE. Such a stimulation was not seen in the presence of TFIIE-DeltaC.

Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions.

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.