Protein-RNA interactions in the active center of transcription elongation complex.

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment.

Protein-protein interactions in eukaryotic transcription initiation: structure of the preinitiation complex.

We have used alanine scanning to analyze protein-protein interactions by human TATA-element binding protein (TBP) within the transcription preinitiation complex. The results indicate that TBP interacts with RNA polymerase II and general transcription factors IIA, IIB, and IIF within the functional transcription preinitiation complex and define the determinants of TBP for each of these interactions. The results permit construction of a model for the structure of the preinitiation complex.

Protein-protein interactions in the rigor actomyosin complex.

Since it has not been possible to crystallize the actomyosin complex, the x-ray structures of the individual proteins together with data obtained by fiber diffraction and electron microscopy have been used to build detailed models of filamentous actin (f-actin) and the actomyosin rigor complex. In the f-actin model, a single monomer uses 10 surface loops and two alpha-helices to make sometimes complicated interactions with its four neighbors. In the myosin molecule, both the essential and regulatory light chains show considerable structural homology to calmodulin. General principles are evident in their mode of attachment to the target alpha-helix of the myosin heavy chain. The essential light chain also makes contacts with other parts of the heavy chain and with the regulatory light chain. The actomyosin rigor interface is extensive, involving interaction of a single myosin head with regions on two adjacent actin monomers. A number of hydrophobic residues on the apposing faces of actin and myosin contribute to the main binding site. This site is flanked on three sides by charged myosin surface loops that form predominantly ionic interactions with adjacent regions of actin. Hydrogen bonding is likely to play a significant role in actin-actin and actin-myosin interactions since many of the contacts involve loops. The model building approach used with actomyosin is applicable to other multicomponent assemblies of biological interest and is a powerful method for revealing molecular interactions and providing insights into the mode of action of the assemblies.

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Receptor-G protein interaction is characterized by cycles of association and dissociation. We present evidence which indicates that during receptor-G protein interaction, the C-terminal tail of the G protein gamma subunit, which is masked in the beta gamma complex, is exposed and establishes high-affinity contact with the receptor. This potential conformational switch provides a mechanism to regulate receptor-G protein coupling. This switch may also be significant for the role of the beta gamma complex in regulation of effector function.

Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex.

Calcium-dependent homotypic cell-cell adhesion, mediated by molecules such as E-cadherin, guides the establishment of classical epithelial cell polarity and contributes to the control of migration, growth, and differentiation. These actions involve additional proteins, including alpha- and beta-catenin (or plakoglobin) and p120, as well as linkage to the cortical actin cytoskeleton. The molecular basis for these interactions and their hierarchy of interaction remain controversial. We demonstrate a direct interaction between F-actin and alpha (E)-catenin, an activity not shared by either the cytoplasmic domain of E-cadherin or beta-catenin. Sedimentation assays and direct visualization by transmission electron microscopy reveal that alpha 1(E)-catenin binds and bundles F-actin in vitro with micromolar affinity at a catenin/G-actin monomer ratio of approximately 1:7 (mol/mol). Recombinant human beta-catenin can simultaneously bind to the alpha-catenin/actin complex but does not bind actin directly. Recombinant fragments encompassing the amino-terminal 228 residues of alpha 1(E)-catenin or the carboxyl-terminal 447 residues individually bind actin in cosedimentation assays with reduced affinity compared with the full-length protein, and neither fragment bundles actin. Except for similarities to vinculin, neither region contains sequences homologous to established actin-binding proteins. Collectively these data indicate that alpha 1 (E)-catenin is a novel actin-binding and -bundling protein and support a model in which alpha 1(E)-catenin is responsible for organizing and tethering actin filaments at the zones of E-cadherin-mediated cell-cell contact.

Evidence that the 50-kDa substrate of brefeldin A-dependent ADP-ribosylation binds GTP and is modulated by the G-protein beta gamma subunit complex.

Brefeldin A, a fungal metabolite that inhibits membrane transport, induces the mono(ADP-ribosyl)ation of two cytosolic proteins of 38 and 50 kDa as judged by SDS/PAGE. The 38-kDa substrate has been previously identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report that the 50-kDa BFA-induced ADP-ribosylated substrate (BARS-50) has native forms of 170 and 130 kDa, as determined by gel filtration of rat brain cytosol, indicating that BARS-50 might exist as a multimeric complex. BARS-50 can bind GTP, as indicated by blot-overlay studies with [alpha-32P]GTP and by photoaffinity labeling with guanosine 5'-[gamma-32P] [beta,gamma-(4-azidoanilido)]triphosphate. Moreover, ADP-ribosylation of BARS-50 was completely inhibited by the beta gamma subunit complex of G proteins, while the ADP-ribosylation of GAPDH was unmodified, indicating that this effect was due to an interaction of the beta gamma complex with BARS-50, rather than with the ADP-ribosylating enzyme. Two-dimensional gel electrophoresis and immunoblot analysis shows that BARS-50 is a group of closely related proteins that appear to be different from all the known GTP-binding proteins.

Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum.

We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding.

Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex.

The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that beta-catenin is the linker protein between E-cadherin and alpha-catenin and that E-cadherin does not bind directly to alpha-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of alpha-catenin are independent binding sites for beta-catenin. In addition to beta-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike beta-catenin, p120 does not bind alpha-catenin in vitro, although a complex of p120 and endogenous alpha-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and alpha-catenin revealed two catenin pools in cell lysates: an approximately 1000- to approximately 2000-kDa complex bound to E-cadherin and an approximately 220-kDa pool that did not contain E-cadherin. Only beta-catenin in the approximately 220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.

Peptide mimicry of the meningococcal group C capsular polysaccharide.

Sequence analysis of the variable regions of the heavy and light chains of the anti-idiotypic antibody 6F9, which mimics the meningococcal group C capsular polysaccharide (MCP), was performed. The immunogenic site on 6F9 responsible for inducing an anti-MCP antibody response was determined by means of sequence and computer model analysis of these data. Complementarity-determining region 3 (CDR3) was found to be unique in that the sequence tract YRY was exposed on the surface. A synthetic peptide spanning the CDR3 domain was synthesized and complexed to proteosomes (meningococcal group B outer membrane protein). Immunizations of BALB/c mice with the peptide-proteosome complex resulted in a significant anti-MCP antibody response. Immunized mice were protected against infection with a lethal dose of Neisseria meningitidis serogroup C.

Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms

The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoletic development and phenotypic variation. BioEssays 25:930-939,2003. (C) 2003 Wiley Periodicals, Inc.

The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

Association behavior of the synthetic protein-lipid complex analogues

Hypercoiling poly(styrene-ALT-maleic anhydride) (PSMA) is known to undergo conformational transition in response to environmental stimuli. This behavior allows it to associate with the phospholipid, 2-dilauryl-SN-glycero-3- phosphocholine (DLPC) to produce nanostructures analogous to lipoproteins. The complex represents a new bio-mimetic delivery vehicle with applications in the cosmetic and pharmaceutical industries. This study investigates, for the first time, the association behavior of PSMA and DLPC through the combination of different analytical techniques. The results indicate that the association is primarily driven by hydrophobic interactions and depends on various factors including the polymer/lipid ratio, the polymer molecular weight and the pH of the aqueous environment. The conformational transition of PSMA leads to the formation of discrete micellar complexes involving anisotropic-to-isotropic lipid phase transformation. As the number of hydrophobic moieties in the polymer is increased, the pH-dependent conformational transition of the polymer plays less important part in achieving this phase transition of the lipid. © (2012) Trans Tech Publications.

The amyloid precursor protein (APP) binds the PIKfyve complex and modulates its function

Phosphoinositides are important components of eukaryotic membranes that are required for multiple forms of membrane dynamics. Phosphoinositides are involved in defining membrane identity, mediate cell signalling and control membrane trafficking events. Due to their pivotal role in membrane dynamics, phosphoinositide de-regulation contributes to various human diseases. In this review, we will focus on the newly emerging regulation of the PIKfyve complex, a phosphoinositide kinase that converts the endosomal phosphatidylinositol-3-phosphate [PI(3)P] to phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2)], a low abundance phosphoinositide of outstanding importance for neuronal integrity and function. Loss of PIKfyve function is well known to result in neurodegeneration in both mousemodels and human patients. Our recent work has surprisingly identified the amyloid precursor protein (APP), the central molecule in Alzheimer s disease aetiology, as a novel interaction partner of a subunit of the PIKfyve complex, Vac14. Furthermore, it has been shown that APP modulates PIKfyve function and PI(3,5)P2 dynamics, suggesting that the APP gene family functions as regulator of PI(3,5)P2 metabolism. The recent advances discussed in this review suggest a novel, unexpected, â-amyloid-independent mechanism for neurodegeneration in Alzheimer s disease.