The ARB (from Latin arbor, tree) project was initiated almost 10 years ago. The ARB program package comprises a variety of directly interacting software tools for sequence database maintenance and analysis which are controlled by a common graphical user interface. Although it was initially designed for ribosomal RNA data, it can be used for any nucleic and amino acid sequence data as well. A central database contains processed (aligned) primary structure data. Any additional descriptive data can be stored in database fields assigned to the individual sequences or linked via local or worldwide networks. A phylogenetic tree visualized in the main window can be used for data access and visualization. The package comprises additional tools for data import and export, sequence alignment, primary and secondary structure editing, profile and filter calculation, phylogenetic analyses, specific hybridization probe design and evaluation and other components for data analysis. Currently, the package is used by numerous working groups worldwide.

A complex array of chaperones and enzymes reside in the endoplasmic reticulum (ER) to assist the folding and assembly of and the disulfide bond formation in nascent secretory proteins. Here we characterize a novel human putative ER co-chaperone (ERdj5)

Basic helix-loop-helix (bHLH) 1 proteins are a class of transcription factors that are important regulators in numerous systems, often involving the control of cell growth and differentiation (1). Most bHLH proteins (with some exceptions) can be broadly classified into two groups based on their patterns of expression. Class A bHLH transcription factors, also called E proteins, are broadly expressed and include the E2A gene products E12/E47 (2) and products of the E2-2/SEF2-1 gene (3, 4). E proteins are capable of forming both homodimers with themselves and heterodimers with cell type-specific class B proteins. This large group of tissue-restricted proteins includes the myogenic proteins (myogenin (5, 6), MyoD (7), MRF4 (8 -10), and Myf5 (11)) and proteins involved in neurogenesis, including MASH2 (12) and NeuroD (13). Functional activity of a class B protein in vivo requires heterodimerization with an E protein, resulting in the commitment of cells to differentiation pathways (14).Calcium plays a crucial role in many cellular processes (15, 16). Its actions are largely mediated through a family of calcium-binding proteins, of which calmodulin is the major calcium sensor. Calmodulin is a highly conserved, ubiquitously expressed protein that is essential for cell growth (17, 18). Calmodulin has four high affinity calcium binding sites called EF-hands, each composed of two ␣-helices connected by a calcium binding loop (19). Upon calcium binding, calmodulin undergoes a conformational change to expose hydrophobic patches (Ref. 20 and references therein), which allows interaction with numerous target proteins and the subsequent activation of signaling pathways.S-100 proteins are other members of the EF-hand protein family that also modulate the activities of various proteins. At least 17 members of the S-100 family have been identified. They vary in their target specificity, cell type distribution, and cell cycle regulation (21). The best studied members of the S-100 family, S-100␣ and S-100␤, have been shown to exist as both homodimers with themselves and heterodimers with each other. Their expression patterns differ; S-100␣ is predominantly found in muscle, whereas S-100␤ is highly expressed in cells within the nervous system (22-26). S-100 proteins are believed to interact with many proteins, and the identification of several common targets with calmodulin suggests that a common structural domain mediates these interactions (27)(28)(29)(30).We have previously shown that calcium-loaded calmodulin can selectively inhibit the DNA binding of E protein homodimers in vitro and that the calcium ionophore ionomycin inhibits their activity in vivo. In contrast, the heterodimers E12/MASH2 and E12/MyoD were either less sensitive or completely resistant (31).Here we identify the protein sequences within the bHLH proteins that determine the differential inhibition by calmodulin. We show that this inhibition is the result of a physical interaction between the DNA binding basic sequence and calmodulin. Both E12 and MyoD basic seq...

The product of the proto-oncogene c-myc influences many cellular processes through the regulation of specific target genes. Through its transactivation domain (TAD), c-Myc protein interacts with several transcription factors, including TATA-binding protein (TBP). We present data that suggest that in contrast to some other transcriptional activators, an extended length of the cMyc TAD is required for its binding to TBP. Our data also show that this interaction is a multistep process, in which a rapidly forming low affinity complex slowly converts to a more stable form. The initial complex formation results from ionic or polar interactions, whereas the slow conversion to a more stable form is hydrophobic in nature. Based on our results, we suggest two alternative models for activation domain/target protein interactions, which together provide a single universal paradigm for understanding activator-target factor interactions.

Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.