Isolation and expression of two genes encoding eukaryotic release factor 1 from Paramecium tetraurelia

Paramecium tetraurelia, like some other ciliate species, uses an alternative nuclear genetic code where UAA and UAG are translated as glutamine and UGA is the only stop codon. It has been postulated that the use of stop codons as sense codons is dependent on the presence of specific tRNAs and on modification of eukaryotic release factor one (eRF1), a factor involved in stop codon recognition during translation termination. We describe here the isolation and characterisation of two genes, eRF1-a and eRF1 b, coding for eRF1 in P. tetraurelia. The two genes are very similar, both in genomic organization and in sequence, and might result from a recent duplication event. The two coding sequences are 1,314 nucleotides long, and encode two putative proteins of 437 amino acids with 98.5% identity. Interestingly, when compared with the eRF1 sequences either of ciliates having the same variant genetic code, or of other eukaryotes, the eRF1 of P. tetraurelia exhibits significant differences in the N-terminal region, which is thought to interact with stop codons. We discuss here the consequences of these changes in the light of recent models proposed to explain the mechanism of stop codon recognition in eukaryotes. Besides, analysis of the expression of the two genes by Northern blotting and primer extension reveals that these genes exhibit a differential expression during vegetative growth and autogamy.     

Transient idling of posttermination ribosomes ready to reinitiate protein synthesis

The fate of ribosomes between termination and initiation during protein synthesis is very basic, yet poorly understood. Here we found that translational reinitiation of the alkaline phosphatase gene occurs in Escherichia coli from an internal methionine codon when the authentic translation is prematurely terminated at a nonsense codon that is within seven codons upstream of the reinitiation codon (which we refer to as "reinitiation window"). Changing the reading frame downstream of the stop codon did not abolish the reinitiation, while inactivating the upstream initiation codon abolished the reinitiation. Moreover, depletion of the ribosome recycling factor (RRF), which disassembles posttermination ribosomes in conjunction with elongation factor G, did not influence the observed reinitiation. These findings suggest that posttermination ribosomes can undergo a transient idling state ready to reinitiate protein synthesis even in the absence of the Shine-Dalgarno (SD) sequence within the reinitiation window by evading disengagement from the mRNA.     

Enzymatic synthesis of a conducting complex of polyaniline and poly(2-arcylamido-2-methyl-1-propanesulfonic ACID) using palm tree peroxidase and its properties

An enzymatic method of producing a conducting polyelectrolyte complex of polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) was developed. Acidic stable peroxidase isolated from royal palm tree (Roystonea regia L.) leaves was used as a catalyst in the oxidative polymerization of aniline at pH 2.8. The synthesis procedure was optimized. Spectroscopic and electrochemical characteristics of nanoparticles of obtained PANI/PAMPS complexes at different pH were studied. It was shown that the acidity of the medium affects their properties.     

Polypeptide release factor eRF1 from Tetrahymena thermophila: cDNA cloning, purification and complex formation with yeast eRF3.

The first cDNA for the translational release factor eRF1 of ciliates was cloned from Tetrahymena thermophila. The coding frame contained one UAG and nine UAA codons that are reassigned for glutamine in Tetrahymena. The deduced protein sequence is 57% identical to human eRF1. The recombinant Tetrahymena eRF1 purified from a yeast expression system was able to bind to yeast eRF3 as do other yeast or mammalian eRF1s as a prerequisite step for protein termination. The recombinant Tetrahymena eRF1, nevertheless, failed to catalyze polypeptide termination in vitro with rat or Artemia ribosomes, at least in part, due to less efficient binding to the heterologous ribosomes. Stop codon specificity and phylogenetic significance of Tetrahymena eRF1 are discussed from the conservative protein feature.     

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro.

Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion. To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis. We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro. We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer.     

Effect of repeated daily electrical stimulation on seizure activity in the rabbit limbic system

The order of appearance, the functional relations, and changes in the epileptiform effects of electrical stimulation of the limbic system (septum, amygdala, hippocampus) were studied. During repeated electrical stimulation regular changes took place in the seizure activity: the duration, frequency, and amplitude of the after-discharges were increased, their polarity was changed, seizures and unsynchronized high-amplitude activity appeared, and the after-discharges were reactivated. The most common variants of the seizure patterns are described. Close correlation was found between the greatest intensity of the after-discharges and the appearance of seizures. Besides a critical number of after-discharges, their parameters and their degree of irradiation also play an important role in the onset of the seizures. It is postulated that the limbic structures may play the role of an organized epileptogenic focus. Facts indicating that the seizure activity in response to direct electrical stimulation of the limbic structures may have a reflex mechanism are presented.     

Oligonucleotide-binding proteins of Drosophila: tissue specificity]

A reaction of native Drosophila proteins with an alkylating oligonucleotide derivative bearing 4-[(N-2-chlorethyl-N-methyl)amino]benzylamine at the 5' terminal phosphate has been investigated. It was found, that the reagent alkylates a few proteins (90, 50, 44, 39, 32 kDa). The modification was organ specific. The labeled 39 kDa protein is present in the ovaries only, while the modified 32 kDa protein is found only in the bulbus.