A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem

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Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

The N-terminal part of Binder of SPerm 5 (BSP5), which promotes sperm capacitation in bovine species is intrinsically disordered

Bovine BSP5 belongs to the Binder of SPerm (BSP) family. BSP5 plays a role in the bovine sperm capacitation by promoting cholesterol and phospholipid efflux. The variable N-terminal part in the BSP proteins is the uncharacterized region with no known function. Full-length, N-terminal part, and individual fibronectin type II domains of bovine BSP5 were cloned, expressed and purified from Escherichia coli. His-S tagged N-terminal part showed large variation in migration on SDS-PAGE in comparison to other constructs. Using mass spectrometry it was demonstrated that the His-S-N-terminal part has the expected molecular mass (13 kDa). The recombinant N-terminal part was sensitive to E. coli endogenous proteases during purification. Denaturing purification involving boiling lysis of cells was carried out, as the protein was thermostable. The His-S-N-terminal part lacked structure as determined by CD analysis. Bioinformatics analyses confirmed that the N-terminal part of bovine BSP5 is intrinsically disordered. In addition, bioinformatics analysis indicated that rabbit BSP and multiple forms of BSP proteins of bovine and equine species possess partially or completely disordered N-terminus. The conservation of disorder at the N-terminus in BSP members belonging to different species suggests a role in biological process such as sperm capacitation and/or sperm-egg interactions.     

Pre-emptive Quality Control of a Misfolded Membrane Protein by Ribosome-Driven Effects

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Application of a photosystem II model for analysis of fluorescence induction curves in the 100 ns to 10 s time domain after excitation with a saturating light pulse

A mathematical model of photosystem II (PSII) events was used to analyze chlorophyll fluorescence transients in the time domain from 100 ns to 10 s after excitation with a saturating 10-ns flash, applied as a part of specialized illumination protocol, using preparations of a thermophilic strain of the unicellular green alga, Chlorella pyrenoidosa Chick (using both intact and diuron-treated cells). Analysis of simulation results has proven that particular attention should be given to flash-induced recombination processes, including nonradiative recombination in PSII, while subsequent charge transfer along the electron transport chain of thylakoid membrane can be adequately described by a single reaction of quinone reoxidation. The PSII model was extended by taking inhibition by diuron of the electron transport in the acceptor side of PSII into account, which allowed simulation of fluorescence induction curves observed in the presence of this inhibitor. The model parameters were determined (stromal pH, rate constants of nonradiative recombination, and the initial reduction state of the quinone pool) which provided adequate simulation of experimentally observed ratios of the maximal and initial fluorescence levels (F _m/F ₀).     

Characterization of the unusual basic proteins of cricket spermatid nuclei on the basis of their molecular weights and amino acid compositions

Typical somatic cell type histones are lost from the nucleus during late spermiogenesis in the house cricket; they are replaced by unusual basic proteins specific to the spermatid. We wish to characterize these proteins because they appear to determine the unusual chromatin structures of the spermatid. Molecular weights of the unusual basic proteins were estimated by chromatographing them on Bio-Gel A 0.5 M agarose columns eluted with 6 M guanidine hydrochloride. Two proteins named TH1 and TH2 have molecular weights in the range spanned by the somatic histones. The molecular weight of TH1 is 17 500 and that of TH2 is 15 500. Three additional spermatid proteins were also analyzed by molecular weight determination. They are called here protamines A, B and C, and they have molecular weights in the range typical of protamines. That of A is 6200, of B is 5500 and of C is 3800. They span the range from the large protamines typical of mammalian sperm to the small protamines of salmonid fish. The molecular weights of the TH proteins were also examined by electrophoresis on SDS-polyacrylamide gels. Amino acid compositions determined for TH1 and TH2 show that both are basic proteins rich in arginine relative to lysine. Their compositions are histone-like, but they appear to be distinct histone types rather than variant forms of the somatic histones.     

In vivo suppression of phytochrome aggregation by the GroE chaperonins in Escherichia coli

When expressed in Escherichia coli, a truncated form of phytochrome (oat PHYA AP3 residues 464-1129) self associates to form a series of products ranging in size from monomers to aggregates of greater than 20 subunits. When these same phytochrome sequences are coexpressed with the chaperonins GroEL and GroES, the truncated phytochrome migrates as a native-like dimer in size exclusion chromatography and no higher-order aggregates were detected. GroEL and GroES inhibition of phytochrome aggregation in E. coli presumably occurs via the suppression of folding pathways leading to incorrectly folded phytochrome.     

The role of the medium in long-range electron transfer

 The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism. Received: 2 January 1997 / Accepted: 6 February 1997