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Peter E Wright - One of the best experts on this subject based on the ideXlab platform.
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induced fit and lock and key recognition of 5 s rna by zinc fingers of Transcription Factor IIIA
Journal of Molecular Biology, 2006Co-Authors: Brian M Lee, Bryan K Clarkson, Maria A Martinezyamout, Jane H Dyson, David A Case, Peter E WrightAbstract:Transcription Factor IIIA (TFIIIA) is a Cys2His2 zinc finger protein that regulates expression of the 5 S ribosomal RNA gene by binding specifically to the internal control element. TFIIIA also functions in transport and storage of 5 S RNA by binding directly to the RNA transcript. To obtain insights into the mechanism by which TFIIIA recognizes 5 S RNA, we determined the solution structure of the middle three zinc fingers bound to the central core of 5 S RNA. Finger 4 utilizes “lock and key” recognition to bind in the widened major groove of the pre-structured RNA loop E motif. This interaction is mediated by direct hydrogen bonding interactions with bases. In contrast, recognition of loop A, a flexible junction of three helices, occurs by an induced fit mechanism that involves reorganization of the conserved CAUA motif and structuring of the finger 5–finger 6 interface to form a complementary RNA binding surface.
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identification of a minimal domain of 5 s ribosomal rna sufficient for high affinity interactions with the rna specific zinc fingers of Transcription Factor IIIA
Journal of Molecular Biology, 1999Co-Authors: Laura Neely, Brian M Lee, Peter E WrightAbstract:Abstract Transcription Factor IIIA of Xenopus laevis serves a dual function during oogenesis and early development: this zinc finger protein binds to the internal promoter element of the 5 S ribosomal RNA genes and acts as a positive Transcription Factor; additionally, the protein functions in 5 S RNA storage. The central four zinc fingers (zf4–7) of the nine-finger protein have been shown to bind 5 S rRNA with comparable or higher affinity than the full-length protein. The role of finger seven in binding affinity has been examined by deletion analysis. A zf4–6 protein binds 5 S RNA with about a sevenfold reduction in binding affinity, compared to zf4–7. The effect of non-specific competitor DNA on binding affinities of the zinc finger peptides was examined and found to have a significant effect on the measured affinities of these peptides for full-length and truncated versions of 5 S RNA. The interaction of zf4–6 with full-length 5 S RNA was far more sensitive to non-specific competitor concentration than was the zf4–7:5 S RNA interaction, suggesting that finger seven contributes to both affinity and specificity in this protein:RNA interaction. In order to map zinc finger binding sites on the 5 S RNA molecule, we generated truncated versions of the RNA and tested these molecules for their binding affinities with zf4–7 and zf4–6. Previous studies showed that a 75 nucleotide long RNA, comprising loop A, helix II, helix V, region E and helix IV, bound zf4–7 with high affinity. Selection and amplification binding assays (selex) have now been used to generate smaller high-affinity binding RNAs. We find that a 55 nucleotide long RNA, comprising loop A, helix V, region E and helix IV, but lacking helix II, retains high affinity for zf4–6. These data are consistent with the proposal that fingers 4–6 bind this central core of 5 S RNA and that finger seven binds the helix II region.
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assessment of major and minor groove dna interactions by the zinc fingers of xenopus Transcription Factor IIIA
Nucleic Acids Research, 1996Co-Authors: Steven J Mcbryant, Karen R Clemens, Benjamin Gedulin, Peter E WrightAbstract:Zinc finger proteins of the Cys2His2 class are DNA sequence-specific Transcription Factors. Previous structural studies of zinc finger protein-DNA complexes have shown that amino acids in the finger tip and alpha-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein Transcription Factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1-3 and 7-9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that a polypeptide containing zinc fingers 1-5 (zf1-5) binds oligonucleotides with modifications in the major groove of the finger 4 binding site with wild-type affinity. Mutagenesis and binding site selection studies were performed to determine whether high affinity DNA binding by zf1-5 requires a particular sequence in the binding site for finger 4. Several mutations in this region of the 5S gene reduced the DNA-binding affinity of zf1-5; however, selection and amplification binding assays did not recover the wild-type finger 4 binding site sequence from a pool of mixed sequence oligonucleotides. Rather, a purine-rich sequence on the top strand was highly selected within the finger 4 binding site. We suggest that high affinity DNA binding by zinc finger 4 may be dictated by a sequence-specific DNA structure rather than by a unique DNA sequence. Deletion of finger 4 from zf1-5 results in a protein with poor binding affinity, demonstrating the importance of finger 4 in proper alignment of neighboring fingers with the DNA, and/or the importance of correct protein-protein interactions between fingers.
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Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus Transcription Factor IIIA
1996Co-Authors: Steven J Mcbryant, Karen R Clemens, Benjamin Gedulin, Peter E WrightAbstract:Zinc finger proteins of the Cys2His2 class are DNA sequence-specific Transcription Factors. Previous structural studies of zinc finger protein–DNA complexes have shown that amino acids in the finger tip and α-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein Transcription Factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1–3 and 7–9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that
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1h 15n and 13c resonance assignments for the first three zinc fingers of Transcription Factor IIIA
Journal of Biomolecular NMR, 1994Co-Authors: Xiubei Liao, Karen R Clemens, John Cavanagh, Linda Tennant, Peter E WrightAbstract:The first three zinc fingers (ZF1-3) of Transcription Factor IIIA (TFIIIA) from Xenopus have been shown to contribute the majority of the binding energy to the intact TFIIIA-DNA interaction [Liao et al. (1992) J. Mol. Biol., 223, 857–871]. We have expressed a 92-amino acid polypeptide containing the three N-terminal zinc fingers of TFIIIA. This three-fingered polypeptide has been isotopically labeled with 15N and 13C in E. coli and purified to homogeneity. Assignment of backbone 1H, 15N, aliphatic 1H and 13C and aromatic 1H and 13C resonances of ΔNZF1-3 has been obtained using a combination of single-, double-and triple-resonance multidimensional NMR experiments. The secondary structures for each finger have been determined from NOE connectivities, 3JNHα values and chemical shifts. The results show that each finger folds into a canonical β-sheet-helix zinc finger structural motif, while the linkers adopt an extended structure. The helix between the two histidine ligands in ZF3 is distorted by zinc coordination, to accommodate the presence of four intervening amino acids instead of three as in ZF1 and ZF2.
Ronald R. Breaker - One of the best experts on this subject based on the ideXlab platform.
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A Plant 5S Ribosomal RNA Mimic Regulates Alternative Splicing of Transcription Factor IIIA Pre-mRNAs
2016Co-Authors: Ming C Hammond, Andreas Wachter, Ronald R. BreakerAbstract:Transcription Factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with striking resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs (pre-mRNAs) from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. Since the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing. A large portion of transcripts from diverse eukaryotes exhibit alternative splicing. Between 40-60 % of expressed human genes are conservatively estimated to give rise to multiple splice products1. Alternative splicing events have recently been shown to be prevalent i
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A plant 5S ribosomal RNA mimic regulates alternative splicing of Transcription Factor IIIA pre-mRNAs.
Nature Structural & Molecular Biology, 2009Co-Authors: Ming C Hammond, Andreas Wachter, Ronald R. BreakerAbstract:Transcription Factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with clear resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. As the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing.
Karen R Clemens - One of the best experts on this subject based on the ideXlab platform.
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assessment of major and minor groove dna interactions by the zinc fingers of xenopus Transcription Factor IIIA
Nucleic Acids Research, 1996Co-Authors: Steven J Mcbryant, Karen R Clemens, Benjamin Gedulin, Peter E WrightAbstract:Zinc finger proteins of the Cys2His2 class are DNA sequence-specific Transcription Factors. Previous structural studies of zinc finger protein-DNA complexes have shown that amino acids in the finger tip and alpha-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein Transcription Factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1-3 and 7-9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that a polypeptide containing zinc fingers 1-5 (zf1-5) binds oligonucleotides with modifications in the major groove of the finger 4 binding site with wild-type affinity. Mutagenesis and binding site selection studies were performed to determine whether high affinity DNA binding by zf1-5 requires a particular sequence in the binding site for finger 4. Several mutations in this region of the 5S gene reduced the DNA-binding affinity of zf1-5; however, selection and amplification binding assays did not recover the wild-type finger 4 binding site sequence from a pool of mixed sequence oligonucleotides. Rather, a purine-rich sequence on the top strand was highly selected within the finger 4 binding site. We suggest that high affinity DNA binding by zinc finger 4 may be dictated by a sequence-specific DNA structure rather than by a unique DNA sequence. Deletion of finger 4 from zf1-5 results in a protein with poor binding affinity, demonstrating the importance of finger 4 in proper alignment of neighboring fingers with the DNA, and/or the importance of correct protein-protein interactions between fingers.
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Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus Transcription Factor IIIA
1996Co-Authors: Steven J Mcbryant, Karen R Clemens, Benjamin Gedulin, Peter E WrightAbstract:Zinc finger proteins of the Cys2His2 class are DNA sequence-specific Transcription Factors. Previous structural studies of zinc finger protein–DNA complexes have shown that amino acids in the finger tip and α-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein Transcription Factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1–3 and 7–9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that
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1h 15n and 13c resonance assignments for the first three zinc fingers of Transcription Factor IIIA
Journal of Biomolecular NMR, 1994Co-Authors: Xiubei Liao, Karen R Clemens, John Cavanagh, Linda Tennant, Peter E WrightAbstract:The first three zinc fingers (ZF1-3) of Transcription Factor IIIA (TFIIIA) from Xenopus have been shown to contribute the majority of the binding energy to the intact TFIIIA-DNA interaction [Liao et al. (1992) J. Mol. Biol., 223, 857–871]. We have expressed a 92-amino acid polypeptide containing the three N-terminal zinc fingers of TFIIIA. This three-fingered polypeptide has been isotopically labeled with 15N and 13C in E. coli and purified to homogeneity. Assignment of backbone 1H, 15N, aliphatic 1H and 13C and aromatic 1H and 13C resonances of ΔNZF1-3 has been obtained using a combination of single-, double-and triple-resonance multidimensional NMR experiments. The secondary structures for each finger have been determined from NOE connectivities, 3JNHα values and chemical shifts. The results show that each finger folds into a canonical β-sheet-helix zinc finger structural motif, while the linkers adopt an extended structure. The helix between the two histidine ligands in ZF3 is distorted by zinc coordination, to accommodate the presence of four intervening amino acids instead of three as in ZF1 and ZF2.
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locations of contacts between individual zinc fingers of xenopus laevis Transcription Factor IIIA and the internal control region of a 5s rna gene
Biochemistry, 1992Co-Authors: Karen R ClemensAbstract:A set of mutants of Transcription Factor IIIA (TFIIIA) have been prepared in which successive zinc-finger domains have been deleted from the carboxyl terminus of the protein. These have been analyzed by hydroxyl radical footprinting to map the location of contacts to DNA by individual zinc-finger domains of TFIIIA. The results suggest that the nine zinc fingers of TFIIIA are organized into three DNA-binding domains of three fingers each. The spatial relationship between zinc-finger contacts to 5S DNA suggests that the two domains which interact with either end of the DNA-binding site of TFIIIA (fingers 1-3 and 7-9) have a compact conformation, similar to that exemplified by the zif268 cocrystal structure [Pavletich, N.P., & Pabo, C.O. (1991) Science 252, 809-817]. However, the central domain (fingers 4-6) has a much more extended conformation, following a path nearly parallel to the helix axis and contacting over 20 base pairs of DNA in the center of the binding site of TFIIIA. These results strongly support two recently proposed and radically new models for the TFIIIA/5S DNA complex [Hayes, J.J., & Tullius, T.D. (1992) J. Mol. Biol. 227, 407-417; Clemens, K.R., Liao, X., Wolf, V., Wright, P.E., & Gottesfeld, J.M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)].
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definition of the binding sites of individual zinc fingers in the Transcription Factor IIIA 5s rna gene complex
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Karen R Clemens, Xiubei Liao, Veronica Wolf, Peter E Wright, Joel M GottesfeldAbstract:Abstract A series of polypeptides containing increasing numbers of zinc fingers of Xenopus Transcription Factor IIIA has been generated and binding to the 5S RNA gene internal control region has been studied in order to elucidate the mode of interaction of the individual fingers with DNA. By using a combination of DNase I footprinting, methylation interference, and differential binding to mixtures of DNA fragments differing in length by single base pairs, the binding sites for individual fingers have been defined. These results have led to a model for the interaction of Transcription Factor IIIA with the internal control region in which fingers 1-3 bind in the major groove of the promoter C block, fingers 7-9 bind in the major groove of the A block, and finger 5 binds in the major groove of the intermediate element. Fingers 4 and 6 each bind across the minor groove, spanning these promoter elements.
Ming C Hammond - One of the best experts on this subject based on the ideXlab platform.
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A Plant 5S Ribosomal RNA Mimic Regulates Alternative Splicing of Transcription Factor IIIA Pre-mRNAs
2016Co-Authors: Ming C Hammond, Andreas Wachter, Ronald R. BreakerAbstract:Transcription Factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with striking resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs (pre-mRNAs) from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. Since the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing. A large portion of transcripts from diverse eukaryotes exhibit alternative splicing. Between 40-60 % of expressed human genes are conservatively estimated to give rise to multiple splice products1. Alternative splicing events have recently been shown to be prevalent i
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A plant 5S ribosomal RNA mimic regulates alternative splicing of Transcription Factor IIIA pre-mRNAs.
Nature Structural & Molecular Biology, 2009Co-Authors: Ming C Hammond, Andreas Wachter, Ronald R. BreakerAbstract:Transcription Factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with clear resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. As the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing.
Jeffrey J. Hayes - One of the best experts on this subject based on the ideXlab platform.
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structural features of Transcription Factor IIIA bound to a nucleosome in solution
Molecular and Cellular Biology, 2004Co-Authors: Joseph M Vitolo, Zungyoon Yang, Ravi Basavappa, Jeffrey J. HayesAbstract:Assembly of a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene into a nucleosome greatly restricts binding of the 5S gene-specific Transcription Factor IIIA (TFIIIA) to the 5S internal promoter. However, TFIIIA binds with high affinity to 5S nucleosomes lacking the N-terminal tail domains of the core histones or to nucleosomes in which these domains are hyperacetylated. The degree to which tail acetylation or removal improves TFIIIA binding cannot be simply explained by a commensurate change in the general accessibility of nucleosomal DNA. In order to investigate the molecular basis of how TFIIIA binds to the nucleosome and to ascertain if binding involves all nine zinc fingers and/or displacement of histone-DNA interactions, we examined the TFIIIA-nucleosome complex by hydroxyl radical footprinting and site-directed protein-DNA cross-linking. Our data reveal that the first six fingers of TFIIIA bind and displace approximately 20 bp of histone-DNA interactions at the periphery of the nucleosome, while binding of fingers 7 to 9 appears to overlap with histone-DNA interactions. Molecular modeling based on these results and the crystal structures of a nucleosome core and a TFIIIA-DNA cocomplex yields a precise picture of the ternary complex and a potentially important intermediate in the transition from naive chromatin structure to productive polymerase III Transcription complex.
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histones h2a h2b inhibit the interaction of Transcription Factor IIIA with the xenopus borealis somatic 5s rna gene in a nucleosome
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Jeffrey J. Hayes, Alan P. WolffeAbstract:Abstract A Xenopus borealis somatic 5S RNA gene was assembled with either the complete octamer of histones, (H2A/H2B/H3/H4)2, or the (H3/H4)2 tetramer of histones that comprises the central protein kernel of the nucleosome. Gel-mobility shifts, DNase I protection, and immunoblotting assays demonstrate that the class III Transcription Factor IIIA (TFIIIA) readily interacts with 5S DNA associated with the tetramer but that little or no binding is detected when 5S DNA is associated with the full octamer of histones. Thus, the presence of histones H2A and H2B in the 5S nucleosome significantly inhibits the interaction of TFIIIA with its cognate binding site within the 5S RNA gene. We propose that either the depletion of histones H2A and H2B from preexisting nucleosomes or the staged assembly of chromatin after replication in which a tetramer of histones H3/H4 associates with DNA before histones H2A/H2B will facilitate the binding of Transcription Factors to their cognate DNA sequences.