Release factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies.

Prokaryotic class I release factors (RFs) respond to mRNA stop codons and terminate protein synthesis. They interact with the ribosomal decoding site and the peptidyl-transferase centre bridging these 75 A distant ribosomal centres. For this an elongated RF conformation, with partially unfolded core domains II.III.IV is required, which contrasts the known compact RF crystal structures. The crystal structure of Thermus thermophilus RF2 was determined and compared with solution structure of T. thermophilus and Escherichia coli RF2 by microcalorimetry, circular dichroism spectroscopy and small angle X-ray scattering. The structure of T. thermophilus RF2 in solution at 20 degrees C is predominantly compact like the crystal structure. Thermodynamic analysis point to an initial melting of domain I, which is independent from the melting of the core. The core domains II.III.IV melt cooperatively at the respective physiological temperatures for T. thermophilus and E. coli. Thermodynamic analyses and the X-ray scattering results for T. thermophilus RF2 in solution suggest that the compact conformation of RF2 resembles a physiological state in absence of the ribosome.

Poor detection of low-virulence field strains of L. monocytogenes is related to selective agents in selective media and is unrelated to PrfA.

We have previously shown a relationship between the virulence level of Listeria monocytogenes strains and their detection on PALCAM medium. To account for the fact that only 40% of low-virulence field strains of L. monocytogenes were detected on PALCAM medium compared to 92% on ALOA medium, the detection of virulent and low-virulence strains on decomposed selective ALOA and PALCAM media was compared. This showed that better detection of the strains was not explained by the growth factors added to the ALOA medium. On the other hand, the presence of acriflavine in the PALCAM medium partly explained the delay in detection of the low-virulence strains, while the presence of ceftazidime was related to growth inhibition. However, the effect of these two components was modified when they were combined in the PALCAM medium. As some of these low-virulence strains had an inactive PrfA (the transcriptional activator of the main virulence genes of L. monocytogenes), its role in the poor detection of these low-virulence strains was investigated. However, complementing these strains with the wild-type prfA gene or deleting the prfA gene from a virulent strain suggested that this poor detection was unrelated to PrfA, but was related to their higher susceptibility to the antimicrobial components in the selective media.

A User's Guide for Phase Separation Assays with Purified Proteins.

The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.

Purification and characterization of TTFI, a factor that mediates termination of mouse ribosomal DNA transcription.

Termination of rRNA gene transcription is dependent on an 18-base-pair sequence motif, AGGTCGAC CAG AT TA NTCCG (the Sal box), which is present several times in the spacer region downstream of the 3' end of the pre-rRNA coding region. We report here the purification to molecular homogeneity of a nuclear factor which specifically interacts with the Sal box element. Addition of the isolated protein to S-100 extracts which contain low levels of the Sal box-binding protein and are therefore termination incompetent restores terminating activity, indicating that this protein is a polymerase I-specific transcription termination factor. The purified protein (termed TTFI) has a molecular weight of approximately 105,000 on sodium dodecyl sulfate-polyacrylamide gels. Mild proteolysis generates a relatively protease-resistant core which still specifically recognizes its target sequence. However, the termination activity has been lost, suggesting that the interaction with the DNA and the interaction with the transcription apparatus reside in different protein domains.

Characterization of Ers, a PrfA-like regulator of Enterococcus faecalis.

We have identified a transcriptional regulator, named Ers (for enterococcal regulator of survival), of Enterococcus faecalis, an important opportunistic bacterium commonly recovered from hospitalized patients. Ers is a member of the Crp/Fnr family and is 69% similar to Srv, a PrfA-like regulator of Streptococcus pyogenes implicated in virulence, and is the E. faecalis protein most closely related to PrfA, a positive regulator of virulence genes in Listeria monocytogenes. In an in vivo-in vitro macrophage infection model, the survival of an ers mutant was highly significantly decreased compared with that of the parental strain JH2-2. This mutant was more than 10-fold more sensitive to oxidative challenge by hydrogen peroxide. In order to identify genes whose expression was under Ers control, the RNA levels of 31 likely candidates were measured by real-time quantitative PCR. The results indicate that ers may be autoregulated and that the locus ef0082 appears to be positively regulated by Ers. Nevertheless, mutation of ef0082 did not result in any detectable changes in the survival of the bacterium within murine macrophages.

The concentration of polypeptide chain release factors 1 and 2 at different growth rates of Escherichia coli.

The number of molecules of release factor-1 (RF-1) and release factor-2 (RF-2) per Escherichia coli cell grown at various rates was determined using quantitative Western blotting of total solubilized cell protein. The number of RF-1 molecules per cell increased from 1200 to 4900, and of RF-2 from 5900 to 24,900 as growth rates increased from 0.3 to 2.4 doublings per hour. The cellular concentration of the release factors, and therefore efficient termination of protein synthesis is maintained by the increased expression of both RFs as growth rate increases. The expression of both release factors RF-1 and RF-2 is co-ordinated with that of the rest of the translational apparatus, although the increases are less for RF than that for the ribosomes under the same conditions. A significant proportion of the RF pool was found associated with the ribosome fraction. The percentage of ribosomes containing an RF molecule increased from 21 to 33% as the translational rate increased over the growth rate range. Since the cellular concentration of the release factors and their specific activity does not vary significantly with growth rate, this can not provide for an increase in the rate at any of the steps of termination. The postulated strong stop signals, UAAU and UAAG, in genes that are highly expressed at fast growth rates, may result in an increase in the termination rate as a consequence of increased efficiency of decoding by RFs.

Translation termination factor aRF1 from the archaeon Methanococcus jannaschii is active with eukaryotic ribosomes.

Class-1 translation termination factors (release factors (RFs)) from Eukarya (eRF1) and Archaea (aRF1) exhibit a high degree of amino acid sequence homology and share many common motifs. In contrast to eRF1, function(s) of aRF1 have not yet been studied in vitro. Here, we describe for the first time the cloning and expression in Escherichia coli of the gene encoding the peptide chain RF from the hyperthermophilic archaeon Methanococcus jannaschii (MjaRF1). In an in vitro assay with mammalian ribosomes, MjaRF1, which was overproduced in E. coli, was active as a RF with all three termination codon-containing tetraplets, demonstrating the functional resemblance of aRF1 and eRF1. This observation confirms the earlier prediction that eRF1 and aRF1 form a common structural-functional eRF1/aRF1 protein family, originating from a common ancient ancestor.

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.

Purification and properties of the polypeptide chain release factor (RF-4) from an extreme thermophile, Thermus thermophilus HB8.

The polypeptide chain release factor 1 (RF-1) has been purified from an extreme thermophile, Thermus thermophilus HB8. The purification procedure included steps of aqueous two-phase partition, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, Sephadex G-150, and CM-Sephadex. The preparation was more than 90% pure as judged by polyacrylamide gel electrophoresis. The specific activity was about 3.3 pmol of formyl-[3H]-methionine released in 1 min at 25 degrees C per microgram of protein under the standard assay conditions using 4 pmol of the initiation complex and 1 nmol of UpApG. The molecular weight as determined by gel filtration on Sephadex G-150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58,000 and 45,000, respectively. As expected, the factor was extremely heat-stable, 50% of its activity remaining after incubation for 5 min at 84 degrees C. Several properties of the reaction catalyzed by RF-1 are also described.

Aminoterminal extension peptides from type I procollagen normalize excessive collagen synthesis of scleroderma fibroblasts.

Fibroblasts derived from a skin biopsy of a patient with scleroderma in the sclerotic stage were shown to have a higher rate of DNA synthesis, and to synthesize more collagen than fibroblasts from a healthy control. The addition of procollagen peptides to the culture medium of scleroderma fibroblasts almost normalized the collagen synthesis. This observation indicates that the mechanism for the regulation of collagen synthesis by feed back inhibition of prollagen peptides is functioning in this disease. It is suggested that the level of biologically active procollagen peptides is lowered.

Unequivocal single-molecule force spectroscopy of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are predicted to represent about one third of the eukaryotic proteome. The dynamic ensemble of conformations of this steadily growing class of proteins has remained hardly accessible for bulk biophysical techniques. However, single-molecule techniques provide a useful means of studying these proteins. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is one of such techniques, which has certain peculiarities that make it an important methodology to analyze the biophysical properties of IDPs. However, several drawbacks inherent to this technique can complicate such analysis. We have developed a protein engineering strategy to overcome these drawbacks such that an unambiguous mechanical analysis of proteins, including IDPs, can be readily performed. Using this approach, we have recently characterized the rich conformational polymorphism of several IDPs. Here, we describe a simple protocol to perform the nanomechanical analysis of IDPs using this new strategy, a procedure that in principle can also be followed for the nanomechanical analysis of any protein.

C-terminal 76 amino acids of eRF3 are not required for the binding of release factor eRF1a from Euplotes octocarinatus.

Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRF1-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation. In the present study, the C-terminal of Euplotes octocarinatus eRF3 (eRF3C) and truncate eRF3C lacking 76 amino acids in C-terminal (eRF3Ct) were expressed in Escherichia coli. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were purified by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides interacted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRF1a in Euplotes octocarinatus.

Termination of translation in eukaryotes.

Termination of translation is governed in ribosomes by polypeptide chain release factors (pRF and eRF in prokaryotes and eukaryotes, respectively). In prokaryotes, three pRF have been indentified and sequenced, while in eukaryotes, only a single eRF has been identified to date. Recently, we have characterized a highly conserved protein family called eRF1. At least, human and Xenopus laevis proteins from this family are active as eRFs in the in vitro assay with any of the three stop codons. No structural similarity has been revealed between any of the three pRFs and eRF1 family. Furthermore, GTP-binding motifs have not been revealed, although translation termination in eukaryotes is a GTP-dependent process. We have demonstrated that in eukaryotes a second eRF exists in addition to eRF1, called eRF3. The eRF3 family has two features in common: presence of GTP-binding motifs and high conservation of the C-terminal domain structure. The C-terminal domain of the X. laevis eRF3 has no RF activity although it stimulates the eRF1 activity considerably at low concentration of the stop codons, conferring GTP dependence to the termination reaction. Without eRF3, the eRF1 activity is entirely GTP independent. Some features of X. laevis eRF3 (C-terminal domain) resemble those of pRF3. The newly identified eRF1 and eRF3 are structurally conserved and distinct from the respective pRF1/2 and pRF3 proteins, pointing to the possibility of different evolution of translation termination machinery in prokaryotes and eukaryotes. Bipartition of the translation termination apparatus probably provides high rate and accuracy of translation termination.

Isolation and structural analysis of the circulating human cardiodilatin (alpha ANP).

A new method was applied to isolate a polypeptide hormone from human blood. The polypeptides from 1,000 1 of hemofiltrate with a molecular weight lower than 20 kDaltons were adsorbed to 2.5 kg alginic acid, then eluted, precipitated, and desalted on a G-25 Sephadex column, thus obtaining a crude lyophilised plasma polypeptide extract. These polypeptides were further submitted to ion-exchange chromatography. Thereafter, two steps of HPLC were carried out to purify a distinct polypeptide which was the circulating form of cardiodilatin (CDD) in this case. The amino acid analysis, C-terminal enzymatic cleavage by carboxypeptidase A, and sequence analysis showed that the only form of circulating cardiodilatin is the 28 amino acid residue containing molecule, cardiodilatin-99-126 cleaved from the C-terminus of cardiodilatin-126 and identical with alpha-ANP (alpha atrial natriuretic polypeptide). Other bioactive molecular forms of the polypeptide hormones of the cardiodilatin family were not detected in the hemofiltrate. The isolation procedure was followed up by a bioassay using in vitro vascular smooth muscle relaxation.