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Biomedical subjects

C Gualerzi

Publications and source records attributed to C Gualerzi.

At least 55 records · Page 3Linked to original sources
PubMed1977

Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3.

Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes. The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex. The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome. The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction. This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex. Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit. The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.

Escherichia coli↗
PubMed1976

Reactivity of ribosomal sulfhydryl groups in 30S ribosomal subunits of Escherichia coli and 30S-IF-3 complexes.

The reaction of 30S subunits with the SH group reagent N-ethylmaleimide (NEM) causes the loss of approximately 60% of their synethetic activity, but has little or no effect on the ribosomal binding of initiation factor IF-3. The ribosomal binding of this factor, on the other hand, was found to significantly influence the rate and the extent to which several 30S ribosomal proteins react with radioactively labeled NEM when the reaction kinetics of individual ribosomal proteins toward NEM were compared in 30S and 30S-IF-3 complexes. Of the nine 30S ribosomal proteins which react with NEM, proteins S1, S11, S12, and S18 were found to have lower reactivities, while proteins S4 and S21 displayed higher reactivity in the presence of IF-3. The reactivity of proteins S8, S13, and S17, on the other hand, appeared to be influenced little or not at all by the presence of the factor. These results are interpreted as supporting evidence for the premise that the binding of IF-3 results in a conformational change of the 30S subunit.

Binding Sites↗
PubMed1976

Specificity and properties of the destabilization, induced by initiation factor IF-3, of ternary complexes of the 30-S ribosomal subunit, aminoacyl-tRNA and polynucleotides.

Initiation factor IF-3 causes the destabilization of preformed ternary complexes of 30-S ribosomal subunit, codons and aminoacyl-tRNAs or peptidyl-tRNA. This destabilization is dilution-dependent and affects all ternary complexes with the exception of those containing the initiator fMet-tRNA, which remain more resistant to IF-3-induced destabilization under the various conditions studied. Several possible reasons for this specificity have been examined. It was found that the basis for the specificity is not: (a) an intrinsic greater stability of the ternary complexes containing fMet-tRNA, (b) the amoung of aminoacyl-tRNA bound to the ribosome, (c) the conditions under which the ternary complex is made or (d) the formylation of the amino group. On the other hand, the nature of the polynucleotide in response to which the ternary complex is formed was found to influence the amount of aminoacyl-tRan bound to the ribosome, and to some extent the amount of aminoacyl-tRNA which can be relased. The ternary complex containing the mischarged initiator tRNA fVal-tRNAfMet displays greater resistance to the IF-3-induced destabilization than the complex containing fVal-tRNAVal. These results indicate that the specificity of the IF-3 activity is due to the special structural feature of the initiator tRNA molecule and to some extent to the nature of the polynucleotide. The IF-3-induced destabilization of ternary complexes was found to be little affected by variations in reaction conditons, so that this IF-3 activity can be used to measure the stoichiometric binding of IF-3 to the ribosome over a broad range of pH and K+ and Mg2+ concentrations. Several antibiotics have been tested for their capacity to interfere with this reaction; only high concentrations of tetracycline blocked this IF-3 activity.

Anti-Bacterial Agents↗
PubMed1976

The role of 16S rRNA in ribosomal binding of IF-3.

The binding of initiation factor IF-3 to Escherichia coli 30S ribosomal subunits has been found to be inhibited by rRNA ligands such as ethidium bromide, polyamines, and monovalent alkali metals. The order of effectiveness of the polyamines (spermine greater than spermidine greater than putrescine) and alkali metals (Li+ greater than Na+ greater than K+) in inhibiting the ribosomal binding of IF-3 parallels their degree of affinity for the RNA. Furthermore, the binding of IF-3 to 30S subunits chemically modified by photooxidation with rose bengal, nitration with tetranitromethane, and reaction with kethoxal, monoperphthalic acid, and p-chloromercuribenzoic acid was studied. Results obtained after the direct treatment of the 30S subunits with the above chemical reagents or upon reconstitution of 30S particles having a modified rRNA or ribosomal proteins indicate that the IF-3 binding site is preferentially lost when the rRNA becomes modified. It was found that IF-3 could bind normally to 30S subunits lacking protein S1 or proteins S11, S12, S19, and S21 (and perhaps S14) which had been cross-linked to IF-3 in other laboratories.

Bacterial Proteins↗
PubMed1975

The sex-factor-dependent exclusion of coli virus T7.

The cause of T7 exclusion by the F episome was investigated. Extracts from neither normal nor infected F+ cells contained an inhibitor of gene expression in vitro. The protein synthesizing systems prepared in vitro from these cells supported T7 early and late protein synthesis with normal efficiency. The content of translational initiation factors in F- and F+ cells, both noninfected and infected, was almost identical. The episome-dependent block of T7 gene expression was observed only in intact cells and detailed kinetics of gene expression in vivo revealed a stop of all transcription and translation at or just before 11 min after T7 infection. The mechanism of F+-dependent T7 exclusion involves both episomal and viral gene products. The data indicate that a T7-induced membrane alteration of the F+ cell membrane leads to cessation of T7 development as well as to the death of the host cell ('suicide').

Cell Survival↗
PubMed1974

Effect of initiation factor 3 binding on the 30S ribosomal subunits of Escherichia coli.

Under certain conditions, initiation factor 3 (IF-3) can cause the release of aminoacyl-tRNA bound to 30S ribosomal subunits of E. coli. It is shown that this IF-3-induced aminoacyl-tRNA release cannot be attributed to either nucleolytic attack or competition between IF-3 and aminoacyl-tRNA for the same ribosomal binding site. It was found that the 30S-aminoacyl-tRNA-codon complexes formed in the absence of IF-3 are intrinsically different from those prepared in the presence of IF-3. In the absence of IF-3, the ribosomal binding of aminoacyl-tRNA is a virtually irreversible process, since the bound aminoacyl-tRNA can neither be spontaneously released upon dilution nor exchanged for unbound aminoacyl-tRNA. In the presence of IF-3, the binding of one molecule of IF-3 per 30S ribosome renders the binding of aminoacyl-tRNA reversible upon dilution and promotes exchange between bound and unbound aminoacyl-tRNA. It is suggested that this difference is due to a conformational transition of the 30S ribosomal subunit induced by the binding of IF-3. The possible implications of this finding in relation to the mechanism of action of IF-3 and its functional role in the cell are discussed.

Binding Sites↗