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C Gualerzi

Publications and source records attributed to C Gualerzi.

At least 37 records · Page 2Linked to original sources
PubMed1983

Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study.

The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1). The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex. The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.

Escherichia coli↗
PubMed1983

Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites.

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.

Escherichia coli↗
PubMed1983

High resolution 1H-n.m.r. study of the interaction between initiation factor IF1 and 30S ribosomal subunits.

Interaction between Escherichia coli translational initiation factor IF1 (mol. wt. 8119) and 30S ribosomal subunits was followed by high resolution 1H-n.m.r. spectroscopy. Upon gradual addition of increasing yet largely sub-stoichiometric amounts of biologically active deuterated 30S ribosomal subunits, selective line broadenings and chemical shift changes were observed against the background of the gradual disappearance of the whole spectrum. At the highest 30S:IF1 ratio attained (0.25), all the resonance lines were broadened beyond meaningful detection. This behaviour, which can be partly reversed by increasing the ionic strength and/or the temperature, is due to the interaction between IF1 and the 30S ribosomal subunits, and can be explained by the existence of a medium-fast exchange dynamics between free and bound factor. The selective effects observed during titration with 30S ribosomal subunits shed some light on the mode of interaction of IF1 with 30S ribosomal subunits. At least one of the two His residues of the factor appears to be involved in the binding, since it undergoes a low-field change of chemical shift and becomes totally immobilized in the IF1-30S complex. Also strongly implicated in the interaction with 30S are more than one Ser and Arg residue and probably one lysine. Additional effects of the interaction of IF1 with ribosomes are a drastic reduction in the intensity of the ring current upfield shifted methyl resonances and mobilization of a previously rotationally hindered phenylalanine ring.

Escherichia coli↗
PubMed1983

Failure of metoclopramide to affect thyrotropin concentration in the term human fetus.

Metoclopramide (MET), a potent dopamine receptor-blocking drug, or saline was administered to 125 term pregnant women at various time intervals (5-412 min) before delivery. Maternal serum was obtained before and after MET injection. Cord blood was obtained at delivery in MET-treated and saline-treated (control group) women. No significant changes in serum TSH, T4, T3, or rT3 concentrations were observed in maternal or cord blood after MET administration. These results suggest that, in contrast to euthyroid nonpregnant women and men, MET administration does not induce a rise in serum TSH concentration in term pregnant women or in the term fetus. Thus, the dopaminergic inhibitory effect on anterior pituitary TSH secretion may not be an important factor in TSH regulation during pregnancy or in the fetus, or the dose of MET employed may be unable to overcome the dopamine inhibitory effect.

Female↗
PubMed1982

Structure-function relationships in Escherichia coli translational elongation factor G: modification of lysine residues by the site-specific reagent pyridoxal phosphate.

Translational elongation factor G (EF-G) of Escherichia coli was modified with the selective, site-specific lysine reagent pyridoxal phosphate (PLP). The reaction results in the modification of a maximum of 12 lysine residues, one of which is essential for guanosine 5'-triphosphate (GTP) binding and whose modification is inhibited by the presence of GTP. Formation of a reversible adduct between 2,3-butanedione and an essential arginine similarly located in the GTP binding site [Rohrbach, M.S., & Bodley, J. W. (1977) Biochemistry 16, 1360-1363] also protects EF-G from PLP inactivation, suggesting that these two residues are spatially close to each other in the native factor. The essential lysine residue was found in the trypsin-resistant fragment T4 (Mr 41 000). In addition to the lysine essential for GTP binding, at least one further lysine was found to be important for EF-G function, since GTP-protected, PLP-modified EF-G molecules fully competent in binding to 50S ribosomal subunits showed decreased activity in 50S- and 70S-dependent GTP hydrolysis. It is likely that a PLP-modified lysine impairs the interaction of the factor with 30S ribosomal subunits and/or a conformational change of the factor required for the hydrolysis of GTP.

Escherichia coli↗
PubMed1981

Structure-function relationship in Escherichia coli initiation factors. Identification of a lysine residue in the ribosomal binding site of initiation factor by site-specific chemical modification with pyridoxal phosphate.

Incubation of Escherichia coli initiation factor 3 (IF3) with pyridoxal phosphate (PLP) followed by reduction with sodium borohydride resulted in the selective modification and inactivation of this protein. The ribosomal-binding site (RNA-binding site) of IF3 is the target of PLP modification, since (a) the phosphate residue of PLP is required for inactivation; (b) RNA as well as synthetic polynucleotides (especially guanine-containing one) protect IF3 from inactivation; and (c) 30 S, but not 50 S ribosomal subunits, protect IF3 from PLP modification and from inactivation. The incorporation of PLP into IF3 occurred exclusively at lysine residues by reduction of the Schiff bases yielding epsilon-(5'-phosphopyridoxyl)lysine. The PLP-modified lysines were identified by amino acid analysis and sequencing of the PLP-modified peptides. Out of the 20 lysines of the factor, only Lys 2, Lys 5, Lys 99, Lys 112, Lys 166, and an unidentified Lys of the central cluster of the molecule (Lys 86, 87, 90, 91, 96) were found to be modified to varying degrees. The incorporation of 3 to 4 mol of PLP/mol of IF3 is accompanied by a substantial (greater than or equal to 80%) inactivation of this protein; the loss of activity follows apparent first order kinetics, and the inactivation results from the modification of just 1 Lys residue. This essential Lys residue was identified by various criteria to be Lys 112. The identification of an "active region" in the IF3 molecule is emerging from this as well as from other chemical modification studies.

Amino Acids↗
PubMed1979

Identification of the amino acid residues of proteins S5 and S8 adjacent to each other in the 30 S ribosomal subunit of Escherichia coli.

When Escherichia coli 30 S ribosomal subunits are reacted with protein-protein bifunctional reagents, a number of protein pairs as well as aggregates containing three or more ribosomal proteins are formed. In the present study we have purified one of the protein pairs obtained by reaction of 30 S ribosomal subunits with either radioactive or nonradioactive dimethylsuberimidate. Following molecular weight determination and ammonolysis, the pair was shown to consist of ribosomal proteins S5 and S8. The "native" structure of the complex was surmised from its capacity to be reconstituted into a biologically active 30 S ribosomal subunit. From peptide maps and primary structure determination of various peptides it was demonstrated that the cross-linking bond between ribosomal proteins S5 and S8 involves primarily the residues Lys-93 of protein S8 and the COOH-terminal lysine (Lys-166) of ribosomal protein S5. This result is substantiated by the finding that a mutant carrying an altered S5 lacking the COOH-terminal lysine yields a greatly reduced amount of S5-S8 cross-link. In addition to the points of cross-linking it was found that Lys-30, Lys-68, and Lys-86 of S8 and Lys-5 of S5 react with dimethylsuberimidate, indicating that these residues are available for reaction and suggesting their topographical localization on the ribosomal surface.

Amino Acid Sequence↗