Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy

Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional (15N,1H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected. Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and a-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain. The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor

Distribution of selected trace elements in the major fractions of donkey milk

The aim of this study was to evaluate the concentrations of Zn, Cu, Mn, Se, Mo, Co, Li, B, Ti, Cr, Rb, Sr, Cd, and Pb in donkey milk and their distribution in major milk fractions (i.e., fat, casein, whey proteins, and aqueous phase). Individual milk samples were provided by 16 clinically healthy lactating donkeys. Subsequent centrifugation, ultracentrifugation, and ultrafiltration were carried out to remove fat, casein, and whey proteins to obtain skim milk, a supernatant whey fraction, and the aqueous phase of milk, respectively. Concentrations of the elements were measured in whole milk and fractions by inductively coupled plasma-mass spectrometry, and the concentrations associated with fat, casein, and whey proteins were then calculated. The effect of removal of fat, casein, and whey proteins was determined by repeated-measures ANOVA. The fat fraction of donkey milk carried a small (∼4.5% to 13.5%) but significant proportion of Mo, Co, Ti, Cr, and Sr. The casein fraction in donkey milk carried almost all milk Zn, a majority of Cu and Mn, and most of Mo, Ti, and Sr. Relevant proportions, between 20% and 36%, of Se, Co, and Cr were also associated with caseins. The majority of Se, Co, Li, B, Cr, and Rb, and relevant proportions of Mn, Mo, Ti, and Sr were found in soluble form (ultracentrifuged samples) and distributed between whey proteins and the aqueous phase of milk (ultrafiltered samples). Whey proteins in donkey milk carried the majority of milk Se and Co. All Li and B was present in the aqueous phase of milk, which also contained most Rb and Cr, and 17% to 42% of Mn, Se, Mo, Co, Ti, and Sr

Site-directed mutagenesis and NMR spectroscopic approaches to the elucidation of the structure-function relationships in translation initiation factors IF1 and IF3.

The recent developments in the knowledge of the structure and structure-function relationships of prokaryotic initiation factors IF1 and IF3 obtained in our laboratory by site-directed mutagenesis, biochemical and NMR-spectroscopic approaches are discussed

Solar panels as air Cherenkov detectors for extremely high energy cosmic rays

Increasing interest towards the observation of the highest energy cosmic rays has motivated the development of new detection techniques. The properties of the Cherenkov photon pulse emitted in the atmosphere by these very rare particles indicate low-cost semiconductor detectors as good candidates for their optical read-out. The aim of this paper is to evaluate the viability of solar panels for this purpose. The experimental framework resulting from measurements performed with suitably-designed solar cells and large conventional photovoltaic areas is presented. A discussion on the obtained and achievable sensitivities follows.Comment: 6 pages, 8 eps figures included with epsfig, uses espcrc2.sty. Talk given at the Sixth Topical Seminar on Neutrino and Astroparticle Physics, San Miniato, Italy, 17-21 May 199

Sequence-specific recognition of DNA by the C-terminal domain of nucleoid-associated protein H-NS

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage λ CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::λCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::λCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT)with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr109, Arg113, Thr114, and Ala116) belonging to the same protein loop and Glu101. The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex

Search for massive rare particles with the SLIM experiment

The search for magnetic monopoles in the cosmic radiation remains one of the main aims of non-accelerator particle astrophysics. Experiments at high altitude allow lower mass thresholds with respect to detectors at sea level or underground. The SLIM experiment is a large array of nuclear track detectors at the Chacaltaya High Altitude Laboratory (5290 m a.s.l.). The results from the analysis of 171 m$^2$ exposed for more than 3.5 y are here reported. The completion of the analysis of the whole detector will allow to set the lowest flux upper limit for Magnetic Monopoles in the mass range 10$^5$ - 10$^{12}$ GeV. The experiment is also sensitive to SQM nuggets and Q-balls, which are possible Dark Matter candidates.Comment: Presented at the 29-th ICRC, Pune, India (2005

FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments—specifically, the GFP1-10 and GFP11 fragments—we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein

Search for Intermediate Mass Magnetic Monopoles and Nuclearites with the SLIM experiment

SLIM is a large area experiment (440 m2) installed at the Chacaltaya cosmic ray laboratory since 2001, and about 100 m2 at Koksil, Himalaya, since 2003. It is devoted to the search for intermediate mass magnetic monopoles (107-1013 GeV/c2) and nuclearites in the cosmic radiation using stacks of CR39 and Makrofol nuclear track detectors. In four years of operation it will reach a sensitivity to a flux of about 10-15 cm-2 s-1 sr-1. We present the results of the calibration of CR39 and Makrofol and the analysis of a first sample of the exposed detector.Comment: Presented at the 22nd ICNTS, Barcelona 200

Search for strange quark matter and Q-balls with the SLIM experiment

We report on the search for Strange Quark Matter (SQM) and charged Q-balls with the SLIM experiment at the Chacaltaya High Altitude Laboratory (5230 m a.s.l.) from 2001 to 2005. The SLIM experiment was a 427 m$^{2}$ array of Nuclear Track Detectors (NTDs) arranged in modules of $24 \times 24$ cm$^{2}$ area. SLIM NTDs were exposed to the cosmic radiation for 4.22 years after which they were brought back to the Bologna Laboratory where they were etched and analyzed. We estimate the properties and energy losses in matter of nuclearites (large SQM nuggets), strangelets (small charged SQM nuggets) and Q-balls; and discuss their detection with the SLIM experiment. The flux upper limits in the CR of such downgoing particles are at the level of $1.3 10^{-15}$/cm$^{2}$/s/sr (90% CL).Comment: 4 pages, 7 eps figures. Talk given at the 24th International Conference on Nuclear Tracks in Solids, Bologna, Italy, 1-5 September 200

Bulk Etch Rate Measurements and Calibrations of Plastic Nuclear Track Detectors

New calibrations of CR39 and Makrofol nuclear track detectors have been obtained using 158 A GeV Pb (82+) and In (49+) ions; a new method for the bulk etch rate determination, using both cone height and base diameter measurements was developed. The CR39 charge resolution based on the etch-pit base area measurement is adequate to identify nuclear fragments in the interval 7 <= Z/beta <= 49. For CR39 the detection threshold is at REL~50 MeV cm^2/g, corresponding to a nuclear fragment with Z/beta~7. Base cone area distributions for Makrofol foils exposed to Pb (82+) ions have shown for the first time all peaks due to nuclear fragments with Z > 50; the distribution of the etched cone heights shows well separated individual peaks for Z/beta = 78 - 83 (charge pickup). The Makrofol detection threshold is at REL 2700 MeV cm^2/g, corresponding to a nuclear fragment with Z/beta~50.Comment: 11 pages, 5 EPS figures. Submitted to Nucl. Instr. Meth.