A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III.

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402

The O-GlcNAc transferase (OGT) is a unique nuclear and cytosolic glycosyltransferase that contains multiple tetratricopeptide repeats. We have begun to characterize the mechanisms regulating OGT using a combination of deletion analysis and kinetic studies. Here we show that the p110 subunit of the enzyme forms both homoand heterotrimers that appear to have different binding affinities for UDP-GlcNAc. The multimerization domain of OGT lies within the tetratricopeptide repeat domain and is not necessary for activity. Kinetic analyses of the full-length trimer and the truncated monomer forms of OGT suggest that both forms function through a random bi-bi kinetic mechanism. Both the monomer and trimer have similar specific activities and similar K m values for peptide substrates. However, they differ in their binding affinities for UDP-GlcNAc, indicating that subunit interactions affect enzyme activity. The findings that recombinant OGT has three distinct K m values for UDP-GlcNAc and that UDP-GlcNAc concentrations modulates the affinity of OGT for peptides suggest that OGT is exquisitely regulated by the levels of UDP-GlcNAc within the nucleus and cytoplasm.O-GlcNAc is an abundant intracellular posttranslational modification consisting of a single N-acetylglucosamine O-linked to serine/threonine residues. Unlike other carbohydrate modifications, O-GlcNAc is not further modified and is found almost exclusively in the nucleus and cytoplasm. Since it was first described in lymphocytes (1) O-GlcNAc has been found on an ever increasing number of proteins, including RNA polymerase II and its transcription factors, nuclear pore proteins, tumor suppressor proteins, intermediate filaments, viral proteins, and oncoproteins (reviewed in Refs. 2-5). O-GlcNAc is an abundant and dynamic modification exhibiting properties more like phosphorylation than typical N-and O-linked glycosylation (2, 3). The O-GlcNAc modification (termed O-GlcNAcylation) has been suggested to play a direct role in regulating a number of cellular functions including protein synthesis (6, 7), neurofilament assembly (8), and transcription (9 -11). In our laboratory, we have purified and characterized both a UDP-Nacetylglucosamine:peptide N-acetylglucosaminyl-transferase (O-GlcNAc transferase) (12) specific for the attachment of OGlcNAc to proteins and a soluble N-acetyl-␤-D-glucosaminidase (O-GlcNAcase) (13) specific for the removal of O-GlcNAc from proteins. These enzymes may work together to regulate the attachment and removal of O-GlcNAc in response to cellular signals in much the same way that kinases and phosphatases regulate protein phosphorylation.Although significant progress has been made in our understanding of the distribution of O-GlcNAc in the cell, little is known about how the attachment of O-GlcNAc to proteins is regulated. The problem is significant because numerous proteins are O-GlcNAcylated, many at more then one site. A further complexity is added by the lack of a canonical consensus site for the attachment of O-GlcNAc to proteins (4, 5)...

Section: The P110 Subunit Is the Catalytic Subunit Of The Ogt-mentioning

confidence: 99%

mentioning

confidence: 99%

Regulation of a Cytosolic and Nuclear O-GlcNAc Transferase

Kreppel

Hart

1999

389

402

“…In particular, mutations in and around TPR2 have been shown to increase transcription in vivo of a tRNA gene bearing a defective A block (24,25). The same mutations have also been shown to increase transcription of wild-type and mutant templates in vitro by facilitating the recruitment of TFIIIB70 (25).…”

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confidence: 99%

Interactions between the Tetratricopeptide Repeat-containing Transcription Factor TFIIIC131 and Its Ligand, TFIIIB70

Moir

Puglia

Willis

2000

In the transcription of tRNA and 5 S genes by RNA polymerase III, recruitment of the transcription factor (TF)IIIB is mediated by the promoter-bound assembly factor TFIIIC. A critical limiting step in this process is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related factor Brf1p/TFIIIB70. To facilitate biochemical studies of this interaction, we expressed a fragment of TFIIIC131, TFIIIC131-(1-580), that includes the minimal TFIIIB70 interaction domain defined by two-hybrid studies together with adjacent sequences, up to the end of TPR9, implicated in the assembly reaction. TFIIIC131-(1-580) interacts with TFIIIB70 in solution and inhibits the formation of TFIIIB70⅐TFIIIC⅐DNA complexes. In a coupled equilibrium binding assay, the formation of TFIIIC131-(1-580)⅐TFIIIB70 complexes was adequately described by a single-site binding model and yielded an apparent equilibrium dissociation constant of 334 ؎ 23 nM. CD spectroscopy and limited proteolysis experiments defined a well structured and largely protease-resistant core in TFIIIC131-(1-580) comprising part of the hydrophilic amino terminus, TPR1-5, the intervening non-TPR region, and TPR6 -8. CD spectra showed that trifluoroethanol induced significant ␣-helical structure in TFIIIC131-(1-580). A more modest monovalent ion-dependent CD difference was observed in mixtures of TFIIIC131-(1-580) and TFIIIB70, suggesting that formation of the binary complex may proceed with the acquisition of ␣-helicity. Transcription factor (TF)1 IIIC is a large multisubunit complex that is responsible for recruiting the RNA polymerase III (pol III) initiation factor, TFIIIB, to the DNA upstream of the transcription start site of 5 S rRNA, tRNA, and related genes (1, 2). On tRNA and other genes containing A and B block promoter elements, the assembly reaction begins with nucleosome repositioning or displacement to enable high affinity binding of TFIIIC to the promoter (3-5). Interactions with the A block are thermodynamically weaker than those with the B block, and yet simultaneous binding to both sites occurs over a wide range of distances without regard to helical phasing (2). The conformational flexibility of TFIIIC indicated by these studies is also seen during the recruitment of TFIIIB. This is reflected by changes in site-specific photocross-linking of the 131-kDa subunit (TFIIIC131) (6) and by the ability of TFIIIC to co-direct (with the TATA-binding protein (TBP)) the placement of TFIIIB at different locations on the DNA (7).In addition to TFIIIB recruitment, recent studies have shown that TFIIIC has other functions and activities in pol III transcription. Subunits of TFIIIC in yeast (8) and humans (9, 10) have been shown to interact either genetically or biochemically with subunits of the polymerase that have been highly conserved through evolution. These studies suggest possible roles for TFIIIC in polymerase recruitment; pol III holoenzyme stabilization (11); and/or the cycle of elongation, termination, an...

“…Subsequent binding of the TBP and Bdp1 subunits of TFIIIB generates a series of structural changes in Tfc4, Brf1 and DNA that leads to progressively increased accessibility of Brf1 to DNA (8). The protein-protein interactions between TFIIIB subunits, together with DNA bending, generate a highly stable TFIIIB complex, a structure that surrounds and kinetically traps the DNA (9 -12).Tfc4 contains eleven copies of a ubiquitous protein-protein interaction motif known as a tetratricopeptide repeat (13,14).TPR motifs, as the name implies, are typically 34 amino acids in length and fold into two antiparallel ␣-helices (designated A and B, Ref. 15).…”

mentioning

confidence: 99%

“…Tfc4 contains eleven copies of a ubiquitous protein-protein interaction motif known as a tetratricopeptide repeat (13,14).…”

mentioning

confidence: 99%

The Brf1 and Bdp1 Subunits of Transcription Factor TFIIIB Bind to Overlapping Sites in the Tetratricopeptide Repeats of Tfc4

Liao

Willis

Moir

2003

The RNA polymerase III initiation factor TFIIIB is assembled onto DNA through interactions involving the Tfc4 subunit of the assembly factor TFIIIC and two subunits of TFIIIB, Brf1 and Bdp1. Tfc4 contains two arrays of tetratricopeptide repeats (TPRs), each of which provides a binding site for Brf1. Dominant mutations in the ligand binding channel of the first TPR array, TPRs1-5, and on the back side of this array, increase Brf1 binding by Tfc4. Here we examine the biological importance of the second TPR array, TPRs6 -9. Radical mutations at phylogenetically conserved residues in the ligand binding channel of TPRs6 -9 impair pol III reporter gene transcription. Biochemical studies on one such mutation, L469K in TPR7, revealed a defect in the recruitment of Brf1 into TFIIIB-TFIIIC-DNA complexes and diminished the direct interaction between Tfc4 and Brf1. Multicopy suppression analysis implicates TPR9 in Brf1 binding and TPRs7 and 8 in binding to more than one ligand. Indeed, the L469K mutation also decreased the binding affinity for Bdp1 incorporation into TFIIIB-TFIIIC-DNA complexes and inhibited binary interactions between Bdp1 and Tfc4. The Bdp1 binding domain in Tfc4 was mapped to TPRs1-9, a domain that contains both TPR arrays and thus overlaps two of the known binding sites for Brf1. The properties of the L469K mutation identify both Brf1 and Bdp1 as ligands for the second TPR array.The assembly of the initiation factor TFIIIB by TFIIIC is a limiting step in RNA polymerase III (pol III) 1 transcription. Although multiple subunits of both TFIIIB and TFIIIC interact (1-4) TFIIIB assembly is mediated initially by protein-protein interactions between the TFIIIC subunit, Tfc4, and the TFIIIB subunit, Brf1. Biochemical and genetic studies in yeast indicate that the recruitment of Brf1 by TFIIIC is a major limiting step in preinitiation complex assembly (5-7). Subsequent binding of the TBP and Bdp1 subunits of TFIIIB generates a series of structural changes in Tfc4, Brf1 and DNA that leads to progressively increased accessibility of Brf1 to DNA (8). The protein-protein interactions between TFIIIB subunits, together with DNA bending, generate a highly stable TFIIIB complex, a structure that surrounds and kinetically traps the DNA (9 -12).Tfc4 contains eleven copies of a ubiquitous protein-protein interaction motif known as a tetratricopeptide repeat (13,14).TPR motifs, as the name implies, are typically 34 amino acids in length and fold into two antiparallel ␣-helices (designated A and B, Ref. 15). Although no position within the motif is invariant, a pattern of small and large hydrophobic residues defines the loose TPR consensus and provides stacking interactions between adjacent repeats (15-17). Multiple adjacent repeats form a right-handed superhelix in which the inner channel (formed by the A-helices) provides a ligand binding interface (18 -20). The TPRs in Tfc4 are organized into two arrays in the N terminus, TPR1-5 and TPR6 -9 (with five and four repeats, respectively) that are separated by a region of mini...