The Embryonic Enhancer-Binding Protein SSAP Contains a Novel DNA-Binding Domain Which Has Homology to Several RNA-Binding Proteins

103

104

A combination of structural, biochemical, and genetic studies in model organisms was used to infer a cellular role for the human protein (SBDS) responsible for Shwachman-Bodian-Diamond syndrome. The crystal structure of the SBDS homologue in Archaeoglobus fulgidus, AF0491, revealed a three domain protein. The N-terminal domain, which harbors the majority of diseaselinked mutations, has a novel three-dimensional fold. The central domain has the common winged helix-turn-helix motif, and the C-terminal domain shares structural homology with known RNA-binding domains. Proteomic analysis of the SBDS sequence homologue in Saccharomyces cerevisiae, YLR022C, revealed an association with over 20 proteins involved in ribosome biosynthesis. NMR structural genomics revealed another yeast protein, YHR087W, to be a structural homologue of the AF0491 N-terminal domain. Sequence analysis confirmed them as distant sequence homologues, therefore related by divergent evolution. Synthetic genetic array analysis of YHR087W revealed genetic interactions with proteins involved in RNA and rRNA processing including Mdm20/ Nat3, Nsr1, and Npl3. Our observations, taken together with previous reports, support the conclusion that SBDS and its homologues play a role in RNA metabolism.

Section: Resultsmentioning

confidence: 99%

The Shwachman-Bodian-Diamond Syndrome Protein Family Is Involved in RNA Metabolism

Savchenko¹,

Krogan

Cort

et al. 2005

103

104

“…Surprisingly, both EhEBP1 and EhEBP2 contained regions of homology to the RNA recognition motif (RRM), a nucleotide binding motif found in a large group of RNA-binding proteins (24,25). The RRM is also present in several sequence-specific DNA-binding proteins, such as stage-specific activator protein (SSAP) (26). EhEBP1 contained two full copies of the RRM as well as a 54-amino acid region with many acidic and basic residues (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The DNA recognition sites for both of these proteins are AT-rich, and both proteins show sequence-specific binding to both singlestranded and double-stranded DNA. It has been suggested that these AT-rich sites exhibit some single-stranded character in the cell, allowing for interaction between the conserved aromatic residues within the ␤ sheets of the RRM and nucleotides in the recognition site (26). Interestingly, the URE4 motif recognized by EhEBP1 and EhEBP2 is also AT-rich, which might pose problems for sequence-specific recognition in the context of the AT-rich E. histolytica genome.…”

Section: Figmentioning

confidence: 99%

Identification of Two Entamoeba histolyticaSequence-specific URE4 Enhancer-binding Proteins with Homology to the RNA-binding Motif RRM

Schaenman¹,

Gilchrist²,

Mann³

et al. 2001

To study transcriptional regulation in the lower branching eukaryote Entamoeba histolytica, we have identified two sequence-specific DNA-binding proteins that recognize the upstream regulatory element URE4, an enhancer that regulates expression of the Gal/GalNAc lectin heavy subunit gene hgl5. A chromatographic purification of E. histolytica nuclear extracts by gel filtration, cation exchange, and sequence-specific DNA affinity chromatography led to a 700-fold increase in URE4 binding activity and the appearance of two dominant protein species with molecular masses of 28 and 18 kDa. These proteins, termed E. histolytica enhancerbinding proteins 1 and 2 (EhEBP1 and EhEBP2), were sequenced by tandem mass spectroscopy and their corresponding cDNA clones identified. Recombinant EhEBP1 and EhEBP2 were able to bind double-stranded oligonucleotides bearing the URE4 motif in a sequencespecific manner, and antibodies raised against EhEBP1 were able to interfere with the formation of URE4-protein complexes in crude nuclear extracts. Overexpression of EhEBP1 in E. histolytica trophozoites resulted in a 7-fold drop in promoter activity in transiently transfected reporter gene constructs when the URE4 motif was present, confirming its ability to specifically recognize the URE4 motif and suggesting that additional cofactors may be required for transcriptional activation by URE4. Further characterization and identification of binding partners for EhEBP1 and EhEBP2, the first proteins with demonstrated sequence-specific DNA binding activity to be identified in E. histolytica, should provide new insights into transcriptional regulation in this protozoan parasite.

“…Interestingly, in parallel to this work it was shown that HBsu, the HU protein of Bacillus subtilis, specifically binds the Alu domain of a small cytoplasmic RNA (scRNA), a homologue of mammalian signal recognition particle RNA (24). In the eukaryotic field, a growing body of evidence shows that a number of proteins including transcriptional factors containing zinc finger or RNA recognition motifs are able to bind specifically to both DNA and RNA (25)(26)(27)(28)(29)(30)(31)(32)(33).…”

mentioning

confidence: 97%

The Bacterial Histone-like Protein HU Specifically Recognizes Similar Structures in All Nucleic Acids

Balandina¹,

Kamashev²,

Rouvière‐Yaniv

2002

103

HU, a major component of the bacterial nucleoid, shares properties with histones, high mobility group proteins (HMGs), and other eukaryotic proteins. HU, which participates in many major pathways of the bacterial cell, binds without sequence specificity to duplex DNA but recognizes with high affinity DNA repair intermediates. Here we demonstrate that HU binds to doublestranded DNA, double-stranded RNA, and linear DNA-RNA duplexes with a similar low affinity. In contrast to this nonspecific binding to total cellular RNA and to supercoiled DNA, HU specifically recognizes defined structures common to both DNA and RNA. In particular HU binds specifically to nicked or gapped DNA-RNA hybrids and to composite RNA molecules such as DsrA, a small non-coding RNA. HU, which modulates DNA architecture, may play additional key functions in the bacterial machinery via its RNA binding capacity. The simple, straightforward structure of its binding domain with two highly flexible ␤-ribbon arms and an ␣-helical platform is an alternative model for the elaborate binding domains of the eukaryotic proteins that display dual DNA-and RNA-specific binding capacities.The Escherichia coli HU protein is a major component of the bacterial nucleoid (1-3). This small basic histone-like protein that can introduce negative supercoiling into a close circular DNA molecule in the presence of topoisomerase I is highly conserved and found in all bacterial species (4 -7). HU plays a role in DNA replication, recombination, and repair (8 -10). It participates in Mu transposition (11) and regulation of gene transcription (12). HU has been shown to be important for optimal survival of cells in the stationary phase and under various stress conditions (13).HU belongs to the family of architectural nuclear proteins that control DNA topology by introducing bends into doublestranded (ds) 1 DNA and stabilize higher-order nucleoprotein complexes. HU resembles eukaryotic proteins of the high mobility group (HMG) class in its DNA binding properties because it binds dsDNA with low affinity and no sequence specificity. In contrast, it displays high affinity for some altered DNA structures such as junctions, nicks, gaps, forks, and overhangs even under stringent salt conditions (14 -18). The DNA structural motif for HU recognition consists of either two dsDNA modules with propensity to be inclined or one dsDNA module adjacent to a ssDNA binding module (19). X-ray crystallography and NMR studies have established the structure of HU dimer in the absence of DNA (20 -22). The two subunits are intertwined to form a compact ␣-helical hydrophobic core with two extended positively charged ␤-ribbon arms. Our recent studies suggest that HU contacts duplex DNA via the minor groove with its flexible arms, whereas the high affinity binding to its specific binding motif requires an additional contact with the HU body (19). Similar to histones, HU has been shown to bind to poly(U) homopolymer, 2 but the role of this abundant protein in RNA binding was underestimated. Recently we ...