The mechanism for RNA recognition by ANTAR regulators of gene expression

ANTAR proteins are widespread bacterial regulatory proteins that have RNA-binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.     

Domains required for nucleic acid binding activities in chloroplast ribonucleoproteins.

Five ribonucleoproteins (or RNA-binding proteins) from tobacco chloroplasts have been identified to date; each of these contains an acidic N-terminal domain (24-64 amino acids) and two conserved RNA-binding domains (82-83 amino acids). All five ribonucleoproteins can bind to ssDNA and dsDNA but show high specificity for poly(G) and poly(U). Here we present the nucleic acid binding activity of each domain using a series of deletion mutant proteins made in vitro from the chloroplast 29 kDa ribonucleoproteins. The acidic domain does not have a positive effect on binding activities and proteins lacking this domain show higher affinities for nucleic acids than the wild-type proteins. Mutant proteins containing single RNA-binding domains can bind to poly(G) and poly(U), though with lower affinities than proteins containing two RNA-binding domains. The spacer region (11-37 amino acids) between the two RNA-binding domains does not interact with poly(G) or poly(U) by itself, but is required for the additive activity of the two RNA-binding domains. Proteins consisting of two RNA-binding domains but lacking the spacer have the same activity as those containing only one RNA-binding domain. Possible roles for each domain in chloroplast ribonucleoproteins are discussed.     

Statistical analyses of protein sequence alignments identify structures and mechanisms in signal activation of sensor histidine kinases

Statistical analyses of genome sequence‐derived protein sequence data can identify amino acid residues that interact between proteins or between domains of a protein. These statistical methods are based on evolution‐directed amino acid variation responding to structural and functional constraints in proteins. The identified residues form a basis for determining structure and folding of proteins as well as inferring mechanisms of protein function. When applied to two‐component systems, several research groups have shown they can be used to identify the amino acid interactions between response regulators and histidine kinases and the specificity therein. Recently, statistical studies between the HisKA and HATPase‐ATP‐binding domains of histidine kinases identified amino acid interactions for both the inactive and the active catalytic states of such kinases. The identified interactions generated a model structure for the domain conformation of the active state. This conformation requires an unwinding of a portion of the C‐terminal helix of the HisKA domain that destroys the inactive state residue contacts and suggests how signal‐binding determines the equilibrium between the inactive and active states of histidine kinases. The rapidly accumulating protein sequence databases from genome, metagenome and microbiome studies are an important resource for functional and structural understanding of proteins and protein complexes in microbes.     

Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex protein: implications for trans dominance.

The 27-kDa Rex trans-acting protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative proteins. We generated missense mutations in a particular domain of the Rex protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion proteins demonstrated that all of the mutant Rex proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev proteins suggests that this Rex domain is important for oligomerization.     

Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain

There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs.     

RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein.

An RNA-binding protein of 28 kD (28RNP) has been previously isolated from spinach chloroplasts and was found to be required for 3' end processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic and glycine-rich amino terminal domain. Each domain by itself, as well as in combination with other domains, was expressed in bacterial cells and the polypeptides were purified to homogeneity. We have investigated the RNA-binding properties of the different structural domains using UV-crosslinking, saturation binding and competition between the different domains on RNA-binding. It was found that the acidic domain does not bind RNA, but that each of the RNA-binding domains, expressed either individually or together, do bind RNA, although with differing affinities. When either the first or second RNA-binding domain was coupled to the acidic domain, the affinity for RNA was greatly reduced. However, the acidic domain has a positive effect on the binding of the full-length protein to RNA, because the mature protein binds RNA with a better affinity than the truncated protein which lacks the acidic domain. In addition, it was found that a stretch of two or three G residues is enough to mediate binding of the 28RNP, whereas four U residues were insufficient. The implications of the RNA-binding properties of 28RNP to its possible function in the processing of chloroplast RNA is discussed.     

Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

Protein phosphorylation plays a critical role in the regulation and progression of mitosis. >40,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4100 and > 2200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.     

Biologically Active Ultra-Simple Proteins Reveal Principles of Transmembrane Domain Interactions

Specific interactions between the helical membrane-spanning domains of transmembrane proteins play central roles in the proper folding and oligomerization of these proteins. However, the relationship between the hydrophobic amino acid sequences of transmembrane domains and their functional interactions is in most cases unknown. Here, we use ultra-simple artificial proteins to systematically study the sequence basis for transmembrane domain interactions. We show that most short homopolymeric polyleucine transmembrane proteins containing single amino acid substitutions can activate the platelet-derived growth factor β receptor or the erythropoietin receptor in cultured mouse cells, resulting in cell transformation or proliferation. These proteins displayed complex patterns of activity that were markedly affected by seemingly minor sequence differences in the ultra-simple protein itself or in the transmembrane domain of the target receptor, and the effects of these sequence differences are not additive. In addition, specific leucine residues along the length of these proteins are required for activity, and the positions of these required leucines differ based on the identity and position of the central substituted amino acid. Our results suggest that these ultra-simple proteins use a variety of molecular mechanisms to activate the same target and that diversification of transmembrane domain sequences over the course of evolution minimized off-target interactions.     

Membrane topology and predicted RNA-binding function of the 'early responsive to dehydration (ERD4)' plant protein

Functional annotation of uncharacterized genes is the main focus of computational methods in the post genomic era. These tools search for similarity between proteins on the premise that those sharing sequence or structural motifs usually perform related functions, and are thus particularly useful for membrane proteins. Early responsive to dehydration (ERD) genes are rapidly induced in response to dehydration stress in a variety of plant species. In the present work we characterized function of Brassica juncea ERD4 gene using computational approaches. The ERD4 protein of unknown function possesses ubiquitous DUF221 domain (residues 312-634) and is conserved in all plant species. We suggest that the protein is localized in chloroplast membrane with at least nine transmembrane helices. We detected a globular domain of 165 amino acid residues (183-347) in plant ERD4 proteins and expect this to be posited inside the chloroplast. The structural-functional annotation of the globular domain was arrived at using fold recognition methods, which suggested in its sequence presence of two tandem RNA-recognition motif (RRM) domains each folded into βαββαβ topology. The structure based sequence alignment with the known RNA-binding proteins revealed conservation of two non-canonical ribonucleoprotein sub-motifs in both the putative RNA-recognition domains of the ERD4 protein. The function of highly conserved ERD4 protein may thus be associated with its RNA-binding ability during the stress response. This is the first functional annotation of ERD4 family of proteins that can be useful in designing experiments to unravel crucial aspects of stress tolerance mechanism.     

Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain

Mammalian and fungal Diaphanous-related formin homology (DRF) proteins contain several regions of conserved sequence homology. These include an amino-terminal GTPase binding domain (GBD) that interacts with activated Rho family members and formin homology domains that mediate targeting or interactions with signaling kinases and actin-binding proteins. DRFs also contain a conserved Dia-autoregulatory domain (DAD) in their carboxyl termini that binds the GBD. The GBD is a bifunctional autoinhibitory domain that is regulated by activated Rho. Expression of the isolated DAD in cells causes actin fiber formation and stimulates serum response factor-regulated gene expression. Inhibitor experiments show that the effects of exogenous DAD expression are dependent upon cellular Dia proteins. Alanine substitution of DAD consensus residues that disrupt GBD binding also eliminate DAD biological activity. Thus, DAD expression activates nuclear signaling and actin remodeling by mimicking activated Rho and unlatching the autoinhibited state of the cellular complement of Dia proteins.