A glycyl residue at metal ligand position 5 is also an acceptable substitution, as observed with the RING-G domain-containing San1 protein (Dasgupta et al., 2004). A complete list of all RING domain-containing proteins identified can be found in Supplemental Table I. Each type of manipulation would require its own session/day and so each manipulation will “alternate” and thus it is called an “alternating design”. The RING-D domains form a single distinct clade (Supplemental Fig. However, functional studies could uncover unique roles for closely linked genes. However, in contrast with the known DNA-binding zinc finger domains, the RING domain appears to function as a protein-protein interaction domain (Lovering et al., 1993; Borden, 2000). We thank Dr. Richard Gardner (Fred Hutchinson Cancer Research Center, Seattle) for helpful information regarding the Arabidopsis RING-G protein. Western-blot analysis using GST antibodies or His antibodies (Amersham, Buckinghamshire, UK) was also used to confirm the presence and integrity of each fusion protein. The results of the phylogenetic analysis suggest that, though the RING domains share several key features, outside of the conserved metal ligand residues the RING proteins are very distinct. However, no polyubiquitination was observed for the other RING-HCb protein, At3g45555, tested. The TPEN-treated GST-RING-H2 domain-containing protein lost the ability to promote protein ubiquitination (Fig. To help identity regions of the proteins that may interact with substrates, domain analyses of the amino acids outside the RING domain classified RING proteins into 30 different groups. Twenty-two of the 29 GST-RING-H2 proteins analyzed were active in the in vitro assay. Therefore, each potential RING domain was analyzed manually for the presence of, and the distance between, each of the eight zinc-coordinating Cys and/or His residues. In addition to having an Asp at metal ligand position 8, the Rbx/Roc/Hrt RING domain also coordinates a third zinc ion using amino acid residues in the loop between metal ligands 2 and 3 (Chen et al., 2000; Zheng et al., 2002). The RING-RRM combination is present in the yeast CNOT4 E3, which is a component of the CCR4-NOT transcription repressor complex (Albert et al., 2002). It is particularly common in behavioral therapies such as Behavioral Activation , although it is also part of Aaron Beck's Cognitive Therapy . Frequency Graphs display the total frequency of the Targeted Behavior per session. For purification, 100 μL of glutathione agarose beads (Sigma, St. Louis) was added to cleared lysates and incubated for 2 h at 4°C. AGI loci/code of Arabidopsis proteins and accession numbers for all other putative proteins are given. BLAST searches of the translated Arabidopsis genome also identified RING domain-encoding sequences in unannotated regions of the genome (Supplemental Table V). A, Consensus and number of each type of RING domain identified in Arabidopsis. Approximately 5% of the Arabidopsis (Arabidopsis thaliana) proteome is predicted to be involved in the ubiquitination/26S proteasome pathway. Unlike the F-box family, which contains twice as many proteins, the size of the Arabidopsis RING family is comparable to the numbers found in other eukaryotes such as human and Mus musculus, which contain 385 and 305 RING domains, respectively (Semple, 2003). Each glutathione S-transferase (GST)-tagged full-length RING protein was tested in an in vitro ubiquitination assay consisting of recombinant yeast E1, 6×His-tagged recombinant Arabidopsis E2 AtUBC8, and ubiquitin. The majority of these are protein-protein interaction domains, which may function as the substrate-binding domain of the E3 ligase (Table II). Previously described modified RING domains found in Arabidopsis include the RING-v, RING-C2, and RING-G types. also was partially supported by the National Institute of Health Training (grant no. AGI loci/code and RING type for each protein analyzed are indicated above each blot. www.plantphysiol.org/cgi/doi/10.1104/pp.104.052423. The RING genes account for approximately 2% of the predicted protein-encoding genes of Arabidopsis. Obtaining any permission will be the responsibility of the requestor. To define related groups of RING proteins, we analyzed a phylogenetic tree generated using the various types of RING domains. Each type of manipulation would require its own session/day and so each manipulation will “alternate” and thus it is called an “alternating design”. The largest was the RING-H2 type with 241 domains, followed by the RING-HC type with 186 domains (Fig. No complementary DNA (cDNA) sequences are available for these loci (GenBank, July 2004), and reverse transcription (RT)-PCR experiments failed to amplify products that could be sequenced to determine the endogenous splicing pattern (data not shown). These Cys-rich domains typically lacked one or more of the defining characteristics of the canonical RING domains and were not considered further. Functional Analysis is a fundam ental tool of the design process to explore new concepts and define their architectures. Also, between metal ligand positions 6 and 7, the following amino acids are well conserved: C6-x3-[W]-x3-[KG]-x3-4-C7. Conversely, using this same approach, we were unable to detect a complete RING domain at a number of other loci, even though the predicted proteins were related to a particular family of RING proteins (Supplemental Table IV). The amino acid sequence of representative DAR1 (A), DAR2 (B), and DAR3 (C) domains from Arabidopsis are compared to those found in other species. Twenty-five predicted RING-v domains were identified in Arabidopsis. The presence of such a large and diverse number of RING domain-containing proteins that function as ubiquitin E3 ligases suggests that target-specific proteolysis by these E3 ligases is a complex and important part of cellular regulation in Arabidopsis. Ubiquitinated proteins were visualized via western-blot analysis using ubiquitin antibodies.