Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

Kamada, Katsuhiko; Angelis, Jacqueline De; Roeder, Robert G.; Burley, S.K.

doi:10.1073/pnas.051631098

Cited by 49 publications

(68 citation statements)

References 50 publications

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“…The cterFCPbinding site was shown to consist of an exposed hydrophobic groove between ␣-helices H2 and H3 of cterRAP74. This binding site had also been previously postulated based on mutational data (18) mapped on the crystal structure of the zinc-bound cterRAP74 (35). In the NMR structure of the complex, this exposed hydrophobic groove formed by the side chain residues of H2 (L474), H3 (V490, L493, A494, L497, and L500), and S2 (F513) of cterRAP74 binds to the newly formed amphipathic helix H1Ј in cterFCP (Fig.…”

Section: Resultssupporting

confidence: 54%

“…2). It should be noted that an ␣-helix of residues 942-961 in cterFCP had been predicted based on primary sequence analysis in an earlier study (35), and the authors postulated that this helix may be important in binding to cterRAP74. Although there is no evidence for the ␣-helix in the free cterFCP, the predicted helix is fairly close to the 17-residue H1Ј helix we observe in the cterRAP74͞ cterFCP complex.…”

Section: Resultsmentioning

confidence: 99%

“…Similarly to the cter-RAP74-free form (21,35), the cterRAP74 in the complex also forms a winged-helix domain consisting of three consecutive ␣-helices followed by an antiparallel ␤-sheet. The helices are of residues E456 -R465 (H1), T470 -K475 (H2), and S486 -L500 (H3) and are linked by loops of 4 (L1) and 10 (L2) amino acids ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…§ PROCHECK NMR (32) was used to assess the quality of the structures. phobic groove in cterRAP74 (21,35). Because cterFCP is highly acidic, it was hypothesized that the binding of the cterRAP74͞cterFCP complex would most likely involve specific electrostatic interactions.…”

Section: Resultsmentioning

confidence: 99%

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NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1

Nguyen

Abbott

Potempa

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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FCP1 [transcription factor IIF (TFIIF)-associated carboxyl-terminal domain (CTD) phosphatase] is the only identified phosphatase specific for the phosphorylated CTD of RNA polymerase II (RNAP II).The phosphatase activity of FCP1 is enhanced in the presence of the large subunit of TFIIF (RAP74 in humans). It has been demonstrated that the CTD of RAP74 (cterRAP74; residues 436 -517) directly interacts with the highly acidic CTD of FCP1 (cterFCP; residues 879 -961 in human). In this manuscript, we have determined a high-resolution solution structure of a cterRAP74͞cterFCP complex by NMR spectroscopy. Interestingly, the cterFCP protein is completely disordered in the unbound state, but forms an ␣-helix (H1 ; E945-M961) in the complex. The cterRAP74͞cterFCP binding interface relies extensively on van der Waals contacts between hydrophobic residues from the H2 and H3 helices of cterRAP74 and hydrophobic residues from the H1 helix of cterFCP. The binding interface also contains two critical electrostatic interactions involving aspartic acid residues from H1 of cterFCP and lysine residues from both H2 and H3 of cterRAP74. There are also three additional polar interactions involving highly conserved acidic residues from the H1 helix. The cterRAP74͞cterFCP complex is the first highresolution structure between an acidic residue-rich domain from a holoenzyme-associated regulatory protein and a general transcription factor. The structure defines a clear role for both hydrophobic and acidic residues in protein͞protein complexes involving acidic residue-rich domains in transcription regulatory proteins. R NA polymerase II (RNAP II) is a multisubunit enzyme complex that enters the initiation complex with the carboxylterminal domain (CTD) of its largest subunit in an unphosphorylated form (RNAP IIA). The CTD contains a heptapeptide repeat (YSPTSPS) that becomes extensively phosphorylated (RNAP IIO) primarily at serine-2 and -5 during early stages of transcription (1-3). In the last several years, numerous protein kinases have been implicated in the phosphorylation of the CTD (4-8). This phosphorylation of the CTD enables RNAP II to progress from the initiation phase to a stable elongation complex, and the CTD remains extensively phosphorylated throughout the elongation phase of transcription (4-6). After completion of the transcription cycle, this same RNAP II must be in the unphosphorylated form (RNAP IIA) to be recruited back to the initiation complex (9). Therefore, dephosphorylation of the CTD by a phosphatase(s) is essential to generating a form of the polymerase (RNAP IIA) that is capable of reinitiating transcription.FCP1 [transcription factor IIF (TFIIF)-associating component of the CTD phosphatase], the only known RNAP II CTD-specific phosphatase, was originally partially purified from HeLa cell (10) and yeast (11) extracts. From experiments with this partially purified CTD phosphatase, it was determined that both general transcription factors IIB (TFIIB) and IIF (TFIIF) play important roles in regulating its activity (...

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Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1

Nguyen

Abbott

Potempa

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Although not evident from primary sequence, RAP74 and RAP30 subunits are structurally similar, with an intricate series of N-terminal␤-sheets that form a RAP74-RAP30 dimer interface. RAP74 and RAP30 also have similar C-terminal regions with winged helix-turn-helix structures (2,3). The larger size of the human RAP74 subunit can be attributed to an extensive loop rich in Gly, Pro, Ser, Thr, and charged residues separating more structured N-and C-terminal domains (4,5).…”

Section: Tfiifmentioning

confidence: 99%

A Key Role for the α1 Helix of Human RAP74 in the Initiation and Elongation of RNA Chains

Funk¹,

Nedialkov

et al. 2002

Journal of Biological Chemistry

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RNA polymerase II-associating protein 74 (RAP74) is the large subunit of transcription factor IIF (TFIIF), which is essential for accurate initiation and stimulates elongation by RNA polymerase II. Mutations within or adjacent to the ␣1 helix of the RAP74 subunit have been shown to decrease both initiation and elongation stimulation activities without strongly affecting the interactions of RAP74 with the RAP30 subunit or the interaction between TFIIF and RNA polymerase II. In this manuscript, mutations within the ␣1 helix are compared with mutations made throughout the neighboring conserved N-terminal domain of RAP74. Changes within the N-terminal domain include disruptions of specific contacts with the ␣1 helix, which were revealed in the recently published x-ray crystal structure (Gaiser, F., Tan TFIIF1 appears to be an ␣␤ heterodimer of RAP74 and RAP30 subunits, and previous reports that TFIIF may be an ␣ 2 ␤ 2 heterotetramer are not supported by the x-ray crystal structure (1). Although not evident from primary sequence, RAP74 and RAP30 subunits are structurally similar, with an intricate series of N-terminal␤-sheets that form a RAP74-RAP30 dimer interface. RAP74 and RAP30 also have similar C-terminal regions with winged helix-turn-helix structures (2, 3). The larger size of the human RAP74 subunit can be attributed to an extensive loop rich in Gly, Pro, Ser, Thr, and charged residues separating more structured N-and C-terminal domains (4, 5).TFIIF is an RNA polymerase II-specific transcription factor restricted to the eukaryotic kingdom. Fig. 1, therefore, shows an amino acid sequence alignment of the N-terminal regions of several RAP74 homologues spaced throughout eukaryotic evolution. Beneath the alignment, the primary sequence is correlated with regions of secondary structure. Regions of ␣-helix and ␤-sheet are derived from the crystal structure of human TFIIF (1) or else from secondary structure predictions (6 -9) in the regions where structural information is not available. The x-ray crystal structure indicates a unique dimer interface made up of RAP74 ␤-sheets ␤1, ␤2, ␤3, ␤6, ␤7, ␤8, and the corresponding ␤-sheets in RAP30. The ␤4 and ␤5 sheets of RAP74 interact to form the structured base of a loop that diverges from the dimer core. In the crystal structure, the RAP74 ␣1 helix makes intimate contacts with the ␤4 and ␤5 sheets.The ␣1 helix has been shown previously to be highly sensitive to mutation (10, 11). Several single amino acid changes, particularly in hydrophobic residues, cause significant defects in both accurate initiation and elongation. Because of symmetrical effects on initiation and elongation, ␣1 should function by contacting a molecular target common to both processes, which would implicate RNA polymerase II, TFIIF, and DNA as the most likely targets. Mutations in ␣1 do not appear to affect interaction with the RAP30 subunit, and no RAP30 contacts are identified for ␣1 in the crystal structure (1). As far as can be discerned, ␣1 mutations do not have an effect on assembly of gel-shif...

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Protein Interface Zinc Sites: The Role of Zinc in the Supramolecular Assembly of Proteins and in Transient Protein–Protein Interactions

Maret

2004

Handbook of Metalloproteins

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New roles are emerging for zinc in protein quaternary structure and supramolecular assemblies. When zinc bridges the interfaces of proteins via ligands provided by different polypeptide chains, it is either essential for the interaction or it serves mainly as a stabilizing factor. Zinc can cross‐link four, three, or more commonly, two protein monomers. Functions of protein interface zinc sites include catalysis, inhibition of enzymatic or other activity, packaging of proteins for storage, dimerization of proteins, formation of protein/receptor complexes, and construction of molecular scaffolds. Tight control of the availability of zinc and ligand‐centered redox reactions in zinc/thiolate coordination environments of protein interface zinc sites suggest mechanisms for modulating transient protein–protein interactions.

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Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

Cited by 49 publications

References 50 publications

NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1

NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1

A Key Role for the α1 Helix of Human RAP74 in the Initiation and Elongation of RNA Chains

Protein Interface Zinc Sites: The Role of Zinc in the Supramolecular Assembly of Proteins and in Transient Protein–Protein Interactions

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