Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

Potential of genetic algorithms in protein folding and protein engineering simulations.

Genetic algorithms are very efficient search mechanisms which mutate, recombine and select amongst tentative solutions to a problem until a near optimal one is achieved. We introduce them as a new tool to study proteins. The identification and motivation for different fitness functions is discussed. The evolution of the zinc finger sequence motif from a random start is modelled. User specified changes of the lambda repressor structure were simulated and critical sites and exchanges for mutagenesis identified. Vast conformational spaces are efficiently searched as illustrated by the ab initio folding of a model protein of a four beta strand bundle. The genetic algorithm simulation which mimicked important folding constraints as overall hydrophobic packaging and a propensity of the betaphilic residues for trans positions achieved a unique fold. Cooperativity in the beta strand regions and a length of 3-5 for the interconnecting loops was critical. Specific interaction sites were considerably less effective in driving the fold.     

Characterization of stress and protein turnover from protein overexpression in fed-batch E. coli cultures.

A structured kinetic model that accounts for proteolytic degradation due to recombinant protein overexpression is introduced and its performance evaluated by comparison with previously reported fed-batch experimental data. This mathematical model contains an additional pool for a generic key precursor (in our case phenylalanine), an improved IPTG transport term, a phenylalanine transport term, and a variable protein turnover expression that accounts for proteolytic activity. The model predictions concerning proteolytic activity, glucose level, and cell growth are in very good agreement with an amino acid depletion hypothesis. Cultures exposed to greater stress showed higher and/or longer proteolysis, whereas less overall proteolytic activity was observed when the effect of induction was somewhat ameliorated.     

Dual function of protein confinement in chaperonin-assisted protein folding.

The GroEL/GroES chaperonin system mediates the folding of a range of newly synthesized polypeptides in the bacterial cytosol. Using a rapid biotin-streptavidin-based inhibition of chaperonin function, we show that the cage formed by GroEL and its cofactor GroES can have a dual role in promoting folding. First, enclosure of nonnative protein in the GroEL:GroES complex is essential for folding to proceed unimpaired by aggregation. Second, folding inside the cage can be significantly faster than folding in free solution, independently of ATP-driven cycles of GroES binding and release. This suggests that confinement of unfolded protein in the narrow hydrophilic space of the chaperonin cage smoothes the energy landscape for the folding of some proteins, increasing the flux of folding intermediates toward the native state.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.     

Mechanism of dnaB protein action. I. Crystallization and properties of dnaB protein, an essential replication protein in Escherichia coli

Purification and crystallization of dnaB protein from Escherichia coli was performed on a large scale by a simple procedure. From 1.5 kg of cells, 520 mg of dnaB protein were obtained in a 58% yield with a purity greater than 99%. The E. coli cells harbor a high copy-number plasmid carrying the dnaB gene and overproduce the enzyme over 200-fold. The subunit molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 50,000. Based on a native Mr = 290,000 and cross-linking studies that yielded six bands, dnaB protein is judged to be a hexamer, confirming the results of Reha-Krantz, L. J., and Hurwitz, J. (1978) J. Biol. Chem. 253, 4043-4050.     

Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction.

The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.     

Initiation factor protein modifications and inhibition of protein synthesis.

The protein covalent modification state of eucaryotic initiation factors eIF-2 and eIF-4B in HeLa cells was examined after they were exposed to a variety of conditions or treatments that regulate protein synthesis. A few factors (e.g., variant pH and sodium fluoride) altered the phosphorylation state of the initiation factor proteins, but the majority (hypertonic medium, ethanol, dimethyl sulfoxide sodium selenite, sodium azide, and colchicine) had no effect on either protein. While initiation factor phosphorylation may regulate protein synthesis in response to many physiological situations, other pathways can regulate protein synthesis under nonphysiological circumstances.     

Movement and self-control in protein assemblies. Quasi-equivalence revisited.

Purposeful switching among different conformational states exerts self-control in the construction and action of protein assemblies. Quasi-equivalence, conceived to explain icosahedral virus structure, arises by differentiation of identical protein subunits into different conformations that conserve essential bonding specificity. Mechanical models designed to represent the energy distribution in the structure, rather than just the arrangement of matter, are used to explore flexibility and self-controlled movements in virus particles. Information about the assembly of bacterial flagella, actin, tobacco mosaic virus and the T4 bacteriophage tail structure show that assembly can be controlled by switching the subunits from an inactive, unsociable form to an active, associable form. Energy to drive this change is provided by the intersubunit bonding in the growing structure; this self-control of assembly by conformational switching is called "autostery", by homology with allostery. A mechanical model of the contractile T4 tail sheath has been constructed to demonstrate how self-controlled activation of a latent bonding potential can drive a purposeful movement. The gradient of quasi-equivalent conformations modelled in the contracting tail sheath has suggested a workable mechanism for self-determination of tail tube length. Concerted action by assemblies of identical proteins may often depend on individually differentiated movements.     

Synthesis of protein in the pancreas. II. The role of ribonucleoprotein in protein synthesis

1. The ribonucleoprotein of the microsome fraction which sediments at 40,000 R.P.M. as a pellet (and which is referred to as the pellet material) has been studied with reference to its role in protein synthesis in the pancreas. 2. In pellet material nucleic acid and protein form a definite complex as shown by its electrophoretic behavior and unchanging composition under various conditions. 3. Protein of pellet material is not especially rich in the diamino acids. 4. Evidence is brought forward indicating that the protein component of pellet material takes part in the general process of protein synthesis in the cell. (a) The well known correlation between quantity of RNA and rate of protein synthesis in a tissue implicates the protein of the pellet material, for most of the RNA in the pancreas and other tissues is in this material. (b) Uptake of isotopically labelled glycine by the pellet material, confirming results of previous workers, is for short periods greater than in other protein fractions. (c) Comparing the pellet materials of pancreas, liver, and kidney-three tissues with vastly different rates of protein synthesis, in the sequence given-there is a correlation between the quantity of RNA in the pellet and the rate of protein synthesis in the tissue; a similar correlation between quantity of RNA in the pellet material and rate of N(15)-glycine uptake by the protein component of the pellet; and finally, the level of uptake by total protein varies with the tissue and is related to the uptake of N(15)-glycine by protein of the pellet. 5. In the pancreas a distinction can be made between proteins synthesized for secretion and the nucleoprotein of the pellet (not found in the secretion) which, however, takes part in the synthetic process, as shown by the fact that the N(15) uptake by protein of the pellet is increased when the synthesis of digestive enzymes is stimulated by secretion. 6. The time course of N(15) uptake by proteins of the pancreas indicates that pellet protein serves as precursor material in the synthesis of the secretory proteins. 7. Rate of uptake of N(15)-glycine by the purines of RNA of the pellet material is not correlated with uptake by the protein. 8. The uptake of C(14)-alanine by an in vitro system of microsomes + mitochondria is impaired by preincubation of the microsomes with ribonuclease. This is direct experimental evidence for the dependence of protein synthesis upon the presence or intactness of ribonucleic acid in the microsomes.     

Occurrence of P-flavin binding protein in Vibrio fischeri and properties of the protein.

In previous studies involving Photobacterium species we proposed that (i) P-flavin is the product of luciferase, (ii) the physiological function of the lux operon is not to produce light but to produce FP(390) (luxF protein), including its prosthetic group, P-flavin, and (iii) FP(390) reactivates oxidatively inactivated cobalamin-dependent methionine synthase similar to flavodoxin but at relatively high ionic strength. It seems difficult to extend this idea to all luminous bacteria because the luxF gene is not present in the lux operon in Vibrio or Xenorhabdus. But we predicted that a luciferase fragment which binds P-flavin should function like FP(390) in these species. In this study, we isolated P-flavin binding protein from Vibrio fischeri ATCC 7744. The obtained protein was a modified luciferase as expected, in which the beta-subunit was intact but about 25 amino acid residues at the C-terminus of the alpha-subunit were deleted and the prosthetic group was the fully reduced P-flavin. These results strongly support that the physiological function of the lux operon is as described above even in luminous bacteria other than Photobacterium species. We propose that chromophore B reported by Tu and Hastings [Tu, S.-C. and Hastings, J.W. (1975) Biochemistry 14, 1975-1980] is the reduced P-flavin.     

Thiaisoleucine and protein synthesis.

Thiaisoleucine is an isoleucine analogue having the gamma-methylene group of the valerianic carbon chain substituted by a sulphur atom. It has been demonstrated that thiaisoleucine is activated and transferred to tRNAIle by rat liver aminoacyl-tRNA synthetase and inhibits isoleucine incorporation into polypeptides in protein synthesizing systems from rat liver or rabbit reticulocytes, whereas it does not affect either leucine incorporation or ribosome run-off or polypeptide chain elongation rate. All tests were performed in comparison with O-methyl-threonine, an isoleucine analogue with the gamma-methylene group substituted by an oxygen atom. In all the reactions studied, both thiaisoleucine and O-methyl-threonine act as competitive inhibitors of isoleucine. With respect to O-methyl-threonine, thiaisoleucine shows higher activity as an isoleucine inhibitor.