Evaluation and classification of RING-finger domains encoded by the <Emphasis Type="Italic">Arabidopsis</Emphasis> genome

Background

In computational analysis, the RING-finger domain is one of the most frequently detected domains in the Arabidopsis proteome. In fact, it is more abundant in Arabidopsis than in other eukaryotic genomes. However, computational analysis might classify ambiguous domains of the closely related PHD and LIM motifs as RING domains by mistake. Thus, we set out to define an ordered set of Arabidopsis RING domains by evaluating predicted domains on the basis of recent structural data.

Results

Inspection of the proteome with a current InterPro release predicts 446 RING domains. We evaluated each detected domain and as a result eliminated 59 false positives. The remaining 387 domains were grouped by cluster analysis and according to their metal-ligand arrangement. We further defined novel patterns for additional computational analyses of the proteome. They were based on recent structural data that enable discrimination between the related RING, PHD and LIM domains. These patterns allow us to predict with different degrees of certainty whether a particular domain is indeed likely to form a RING finger.

Conclusions

In summary, 387 domains have a significant potential to form a RING-type cross-brace structure. Many of these RING domains overlap with predicted PHD domains; however, the RING domain signature mostly prevails. Thus, the abundance of PHD domains in Arabidopsis has been significantly overestimated. Cluster analysis of the RING domains defines groups of proteins, which frequently show significant similarity outside the RING domain. These groups document a common evolutionary origin of their members and potentially represent genes of overlapping functionality.

     

Local coexpression domains in the genome of rice show no microsynteny with Arabidopsis domains

Plant responses to abiotic stress are determined both by the severity of the stress as well as the metabolic status of the plant. Abscisic acid (ABA) is a key component in integrating these various signals and controlling downstream stress responses. By screening for plants with decreased RD29A:LUC expression, we isolated two alleles, glutamate:glyoxylate transferase1-1 (ggt1-1) and ggt1-2, of a mutant with altered ABA sensitivity. In addition to reduced ABA induction of RD29A, ggt1-1 was altered in ABA and stress regulation of Δ ¹ -pyrroline-5-carboxylate synthase, proline dehydrogenase and 9-cis-epoxycarotenoid dioxygenase 3, which encode enzymes involved in Pro and ABA metabolsim, respectively. ggt1-1 also had altered ABA and Pro contents after stress or ABA treatments while root growth and leaf water loss were relatively unaffected. A light-dependent increase in H₂O₂ accumulation was observed in ggt1-1 consistent with the previously characterized role of GGT1 in photorespiration. Treatment with exogenous H₂O₂, as well as analysis of a mutant in nucleoside diphosphate kinase 2 which also had increased H₂O₂ content but is not involved in photorespiration or amino acid metabolism, demonstrated that the greater ABA stimulation of Pro accumulation in these mutants was caused by altered H₂O₂ content as opposed to other metabolic changes. The results suggest that metabolic changes that alter H₂O₂ levels can affect both ABA accumulation and ABA sensitivity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

Background

Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants.

Results

Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication.

Conclusions

Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.     

The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M(r) protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.     

Identification and mapping of self-assembling protein domains encoded by the Escherichia coli K-12 genome by use of lambda repressor fusions

Self-assembling proteins and protein fragments encoded by the Escherichia coli genome were identified from E. coli K-12 strain MG1655. Libraries of random DNA fragments cloned into a series of lambda repressor fusion vectors were subjected to selection for immunity to infection by phage lambda. Survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. Four hundred sixty-three nonredundant open reading frame-encoded interacting sequence tags (ISTs) were recovered from sequencing 2,089 candidates. These ISTs, which range from 16 to 794 amino acids in length, were clustered into families of overlapping fragments, identifying potential homotypic interactions encoded by 232 E. coli genes. Repressor fusions identified ISTs from genes in every protein-based functional category, but membrane proteins were underrepresented. The IST-containing genes were enriched for regulatory proteins and for proteins that form higher-order oligomers. Forty-eight (20.7%) homotypic proteins identified by ISTs are predicted to contain coiled coils. Although most of the IST-containing genes are identifiably related to proteins in other bacterial genomes, more than half of the ISTs do not have identifiable homologs in the Protein Data Bank, suggesting that they may include many novel structures. The data are available online at http://oligomers.tamu.edu/.     

Genome-wide and structural analyses of pseudokinases encoded in the genome of Arabidopsis thaliana provide functional insights

Protein Kinase-Like Non-Kinases (PKLNKs), commonly known as “pseudokinases”, are homologous to eukaryotic Ser/Thr/Tyr protein kinases (PKs) but lack the crucial aspartate residue in the catalytic loop, indispensable for phosphotransferase activity. Therefore, they are predicted to be “catalytically inactive” enzyme homologs. Analysis of protein-kinase like sequences from Arabidopsis thaliana led to the identification of more than 120 pseudokinases lacking catalytic aspartate, majority of which are closely related to the plant-specific receptor-like kinase family. These pseudokinases engage in different biological processes, enabled by their diverse domain architectures and specific subcellular localizations. Structural comparison of pseudokinases with active and inactive conformations of canonical PKs, belonging to both plant and animal origin, revealed unique structural differences. The currently available crystal structures of pseudokinases show that the loop topologically equivalent to activation segment of PKs adopts a distinct-folded conformation, packing against the pseudoenzyme core, in contrast to the extended and inhibitory geometries observed for active and inactive states, respectively, of catalytic PKs. Salt-bridge between ATP-binding Lys and DFG-Asp as well as hydrophobic interactions between the conserved nonpolar residue C-terminal to the equivalent DFG motif and nonpolar residues in C-helix mediate such a conformation in pseudokinases. This results in enhanced solvent accessibility of the pseudocatalytic loop in pseudokinases that can possibly serve as an interacting surface while associating with other proteins. Specifically, our analysis identified several residues that may be involved in pseudokinase regulation and hints at the repurposing of pseudocatalytic residues to achieve mechanistic control over noncatalytic functions of pseudoenzymes.     

Local coexpression domains of two to four genes in the genome of Arabidopsis

Expression of genes in eukaryotic genomes is known to cluster, but cluster size is generally loosely defined and highly variable. We have here taken a very strict definition of cluster as sets of physically adjacent genes that are highly coexpressed and form so-called local coexpression domains. The Arabidopsis (Arabidopsis thaliana) genome was analyzed for the presence of such local coexpression domains to elucidate its functional characteristics. We used expression data sets that cover different experimental conditions, organs, tissues, and cells from the Massively Parallel Signature Sequencing repository and microarray data (Affymetrix) from a detailed root analysis. With these expression data, we identified 689 and 1,481 local coexpression domains, respectively, consisting of two to four genes with a pairwise Pearson's correlation coefficient larger than 0.7. This number is approximately 1- to 5-fold higher than the numbers expected by chance. A small (5%-10%) yet significant fraction of genes in the Arabidopsis genome is therefore organized into local coexpression domains. These local coexpression domains were distributed over the genome. Genes in such local domains were for the major part not categorized in the same functional category (GOslim). Neither tandemly duplicated genes nor shared promoter sequence nor gene distance explained the occurrence of coexpression of genes in such chromosomal domains. This indicates that other parameters in genes or gene positions are important to establish coexpression in local domains of Arabidopsis chromosomes.     

Genetic identification of an endoproteinase encoded by the adenovirus genome

The temperature-sensitive adenovirus type 2 mutant H2ts1 is defective for polypeptide processing at the non-permissive temperature. We have in the present study mapped the mutation by marker rescue and DNA sequencing techniques: the mutation is a C/T transition located at map co-ordinate 61.1. Previous sequencing studies have identified an uninterrupted translational reading frame in this part of the adenovirus genome, encoding a hypothetical 23 X 10(3) Mr polypeptide. The mutation leads to a proline/leucine substitution in the 23 X 10(3) Mr polypeptide.     

Analysis of electrostatic contributions to the selectivity of interactions between RING-finger domains and ubiquitin-conjugating enzymes

The zinc-coordinated protein motifs known as RING-finger domains, present on a class of ubiquitin ligases (E3's), recruit ubiquitin-conjugating enzymes (E2s), tethering them to substrate proteins for covalent modification with ubiquitin. Each RING-finger domain can recruit different E2s, and these interactions are frequently selective, in that certain RING-finger domains associate preferentially with certain E2s. This selectivity acquires particular biological relevance when the recruited E2s exert specialized functions. We have explored the determinants that specify the presence or absence of experimentally detectable interaction between two RING-finger domains, those on RNF11 and RNF103, and two E2s, UBC13, a specialized E2 that catalyzes ubiquitin chain elongation through Lys63 of ubiquitin, and UbcH7, which mediates polyubiquitylation through Lys48. Through the iterative use of computational predictive tools and experimental validations, we have found that these interactions and their selectivity are partly governed by the combinations of electrostatic interactions linking specific residues of the contact interfaces. Our analysis also predicts that the main determinants of selectivity of these interactions reside on the RING-finger domains, rather than on the E2s. The application of some of these rules of interaction selectivity has permitted us to experimentally manipulate the selectivity of interaction of the RING-finger domain-E2 pairs under study.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

Methylation and demethylation of the Arabidopsis genome

The primary sequence of the genome is broadly constant and superimposed upon that constancy is the postreplicative modification of a small number of cytosine residues to 5-methylcytosine. The pattern of methylation is non-random; some sequence contexts are frequently methylated and some rarely methylated and some regions of the genome are highly methylated and some rarely methylated. Once established, methylation is not static: it can potentially change in response to developmental or environmental cues and this may result in correlated changes in gene expression. Changes can occur passively owing to a failure to maintain DNA methylation through rounds of DNA replication, or actively, through the action of enzymes with DNA glycosylase activity. Recent advances in genetic analyses and the generation of high resolution, genome-wide methylation maps are revealing in unprecedented detail the patterns and dynamic changes of DNA methylation in plants.     

Counting the zinc-proteins encoded in the human genome

Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biological function, or for regulation of their activities or for structural purposes. Genome sequencing projects have provided a huge number of protein primary sequences, but, even though several different elaborate analyses and annotations have been enabled by a rich and ever-increasing portfolio of bioinformatic tools, metal-binding properties remain difficult to predict as well as to investigate experimentally. Consequently, the present knowledge about metalloproteins is only partial. The present bioinformatic research proposes a strategy to answer the question of how many and which proteins encoded in the human genome may require zinc for their physiological function. This is achieved by a combination of approaches, which include: (i) searching in the proteome for the zinc-binding patterns that, on their turn, are obtained from all available X-ray data; (ii) using libraries of metal-binding protein domains based on multiple sequence alignments of known metalloproteins obtained from the Pfam database; and (iii) mining the annotations of human gene sequences, which are based on any type of information available. It is found that 1684 proteins in the human proteome are independently identified by all three approaches as zinc-proteins, 746 are identified by two, and 777 are identified by only one method. By assuming that all proteins identified by at least two approaches are truly zinc-binding and inspecting the proteins identified by a single method, it can be proposed that ca. 2800 human proteins are potentially zinc-binding in vivo, corresponding to 10% of the human proteome, with an uncertainty of 400 sequences. Available functional information suggests that the large majority of human zinc-binding proteins are involved in the regulation of gene expression. The most abundant class of zinc-binding proteins in humans is that of zinc-fingers, with Cys4 and Cys2His2 being the most common types of coordination environment.     

A theory for developmental control by a program encoded in the genome

A new genetic mechanism is proposed to explain the evident order seen in embryonic development. This theory postulates control DNA, a set of genetic elements activated in a specific sequence, one at a time. With each cell division, control of gene expression passes to the next control unit in the series. The complete series of control units would constitute the encoded (and inherited) development program of an organism.