New 16-membered 9-aryl-alkyl oxime derivatives of 5-O-mycaminosyl-tylonolid (OMT) have recently been prepared and were found to exhibit high activity against macrolide-resistant strains. In this study, we show that these compounds do not affect the binding of tRNAs to ribosomes in a cell-free system derived from Escherichia coli and that they cannot inhibit peptidyltransferase, peptidyl-tRNA translocation, or poly(U)-dependent poly(Phe) synthesis. However, they severely inhibit poly(A)-dependent poly(Lys) synthesis and compete with erythromycin or tylosin for binding to common or partially overlapping sites in the ribosome. According to footprinting analysis, the lactone ring of these compounds seems to occupy the classic binding site of macrolides that is located at the entrance of the exit tunnel, whereas the extending alkyl-aryl side chain seems to penetrate deeper in the tunnel, where it protects nucleoside A752 in domain II of 23S rRNA. In addition, this side chain causes an increased affinity for mutant ribosomes that may be responsible for their effectiveness against macrolide resistant strains. As revealed by detailed kinetic analysis, these compounds behave as slow-binding ligands interacting with functional ribosomal complexes through a one-step mechanism. This type of inhibitor has several attractive features and offers many chances in designing new potent drugs.

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [ 14 C]putrescine or [14 C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (K t ϭ 24.58Ϯ 2.24 µM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (K t ϭ 3.63Ϯ 0.43 µM, K′ t ϭ 0.61Ϯ 0.10 µM). From the K i values, it is inferred that N 1 -5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N 1 -acetylspermine, and then N 1 ,N 12-diacetylspermine. N 1 -acetylspermidine and N 8 -acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows:also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N 4 ,N 9 -diacetylspermine, nor N 1 ,N 4 -bis(β-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.Keywords : acylated polyamine ; polyamine uptake; polyamine pool ; cell growth. Polyamines participate in a wide variety of growth pro-tion, two transport systems with different affinities for putrescine have been suggested in E. coli K12 cells grown in low osmolarcesses. Thus, considerable interest has been generated in elucidating the mechanism of polyamine homeostasis in vivo. It is ity medium [16]. Cross-species transfection of DNA from transport-positive cells into transport-negative cells has been used inferred from several lines of evidence that intracellular polyamine concentrations are regulated by the collective effects of a successfully by Igarashi and co-workers ([13] and references catabolic pathway and an efflux mechanism on the one hand, therein), for the isolation of genes encoding polyamine carriers and a biosynthetic pathway and an uptake mechanism on the in E. coli. The proteins encoded by pPT104 and pPT79 operons other [1]. In Escherichia coli cells, this regulation is, to a large constitute the spermidine/spermine-preferential and the putresdegree, achieved by changes in polyamine biosynthesis and up-cine-specific uptake system, respectively [13]. Another putrestake [2, 3].cine-transport system encoded by pPT71 seems to ha...

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

No abstract

In a cell-free system derived from Escherichia coli,various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg 2ϩ (Ac, acetyl). In the absence of factors washable from ribosomes (FWR fraction), mono-acylated or di-acylated analogues of spermine stimulate the binding of AcPhe-tRNA to a lesser degree than spermine, in the order: N 1 -acetylspermine Ͼ N 1 ,N 12 -diacetylspermine Х N 1 ,N 12 -dipivaloylspermine. Also, the above analogues do not show any sparing effect on Mg 2ϩ requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect.In addition, all analogues tested enhance the stability of the ternary complex AcPhe-tRNA-poly(U)-ribosome and the extent of AcPhe-puromycin synthesis, particularly in the absence of the FWR fraction. At the kinetic phase of AcPhe-puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition). Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl-substituent size, charge distribution and on their chain flexibility.Keywords : acylated polyamine; protein synthesis; puromycin reaction; peptidyltransferase.Polyamines are organic polycations that are protonated at physiological pH and can potentially interact with a variety of cellular macromolecules including nucleic acids and proteins [1]. Substantial evidence exists suggesting a significant role of polyamines in regulating the translation process at several levels [1]. Due to its four positive charges, spermine is the most effective of the naturally occurring polyamines both in regulating the ribosomal functions and in decreasing the Mg 2ϩ requirements for protein synthesis. Tabor and Tabor [2] have reported that endogenous spermine does not exist in Escherichia coli. However, small amounts of spermine, about two orders of magnitude lower than those of spermidine, can be detected in E. coli by more sensitive methods [3]. Extensive biochemical studies have focused on the mechanism of spermine biological action in prokaryotic-cell-free systems, since this polyamine consists the parent compound of a large number of synthetic polyamine analogues with antiparasitic and antitumor activity [4Ϫ6].Evidently, spermine markedly stimulates the activity of several in vitro protein-synthesizing systems characterized by low Mg 2ϩ concentrations near those observed in vivo [7Ϫ10]. In a recent report [11], we have demonstrated that, at 6 mM Mg 2ϩ spermine exhibits a concentration-dependent allosteric biphasic