Maria A. Xaplanteri

Biomonitoring of Gulf of Patras, N. Peloponnesus, Greece. Application of a biomarker suite including evaluation of translation efficiency in Mytilus galloprovincialis cells ☆

Specimens of Mytilus galloprovincialis were placed in bow nets and immersed at 3-10 m depth in a clean coastal region (reference area), Itea, and two marine stations along Gulf of Patras, N. Peloponnesus, Greece. One site is near the estuaries of the Glafkos River, which are influenced by local industrial and urban sources (Station 1); the second site, Agios Vasilios, has no evident organic pollution but is enriched in metals, particularly zinc (Station 2). One month after immersion, digestive glands were removed from the mussels and tested for lysosomal membrane stability, metallothionein content, and translational efficiency of ribosomes. In addition, gill cells were isolated and their micronuclei content was determined. Compared with the reference samples, mussels transplanted to Gulf of Patras showed a significant increased lysosomal membrane permeability and metallothionein content, reduced polysome levels, and increased chromosomal damage in relation to the contamination burden of each sampling area. Also, runoff ribosomes from mussels transplanted to Gulf of Patras (that is, ribosomes stripped of endogenous messengers and peptidyl- or/and aminoacyl-tRNAs) were less efficient at initiating protein synthesis in an in vitro-translation system than those prepared from reference samples. The whole set of data suggests that the degree of Gulf of Patras pollution differs between different sites and depends on the proximity of urban sewage and industrial outfalls. In addition, our results emphasize the importance of protein synthesis regulation as a component of the cellular stress response.

Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions.

Polyamine binding to 23S rRNA was investigated, using a photoaffinity labeling approach. This was based on the covalent binding of a photoreactive analog of spermine, N1-azidobenzamidino (ABA)-spermine, to Escherichia coli ribosomes or naked 23S rRNA under mild irradiation conditions. The cross-linking sites of ABA-spermine in 23S rRNA were determined by RNase H digestion and primer-extension analysis. Domains I, II, IV and V in naked 23S rRNA were identified as discrete regions of preferred cross-linking. When 50S ribosomal subunits were targeted, the interaction of the photoprobe with the above 23S rRNA domains was elevated, except for helix H38 in domain II whose susceptibility to cross-linking was greatly reduced. In addition, cross-linking sites were identified in domains III and VI. Association of 30S with 50S subunits, poly(U), tRNAPhe and AcPhe-tRNA to form a post-translocation complex further altered the cross-linking, in particular to helices H11–H13, H21, H63, H80, H84, H90 and H97. Poly(U)-programmed 70S ribosomes, reconstituted from photolabeled 50S subunits and untreated 30S subunits, bound AcPhe-tRNA in a similar fashion to native ribosomes. However, they exhibited higher reactivity toward puromycin and enhanced tRNA-translocation efficiency. These results suggest an essential role for polyamines in the structural and functional integrity of the large ribosomal subunit.

Polyamines affect diversely the antibiotic potency: insight gained from kinetic studies of the blasticidin S and spiramycin interactions with functional ribosomes

The effects of spermine on peptidyltransferase inhibition by an aminohexosylcytosine nucleoside, blasticidin S, and by a macrolide, spiramycin, were investigated in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. Kinetics revealed that blasticidin S, after a transient phase of interference with the A-site, is slowly accommodated near to the P-site so that peptide bond is still formed but with a lower catalytic rate constant. At high concentrations of blasticidin S (>10 × Ki), a second drug molecule binds to a weaker binding site on ribosomes, and this may account for the onset of a subsequent mixed-noncompetitive inhibition phase. Spermine enhances the blasticidin S inhibitory effect by facilitating the drug accommodation to both sites. On the other hand, spiramycin (A) was found competing with puromycin for the A-site of AcPhe-tRNA·poly(U)·70 S ribosomal complex (C) via a two-step mechanism, according to which the fast formation of the encounter complex CA is followed by a slow isomerization to a tighter complex, termed C*A. In contrast to that observed with blasticidin S, spermine reduced spiramycin potency by decreasing the formation and stability of complex C*A. Polyamine effects on drug binding were more pronounced when a mixture of spermine and spermidine was used, instead of spermine alone. Our kinetic results correlate well with cross-linking and crystallographic data and suggest that polyamines bound at the vicinity of the antibiotic binding pockets modulate diversely the interaction of these drugs with ribosomes.

Probing Ribosomal Proteins Capable of Interacting with Polyamines

The ribosome decodes the genetic information, controls the fidelity of codon-anticodon interactions, and catalyzes the peptide bond formation. However, none of these functional properties can be detected in free rRNAs, because ribosomal proteins and ions are required for the attainment of the rRNA proper tertiary structure (Burma et al. 1985). Despite the fact that the binding sites of ribosomal proteins in rRNA are well characterized, our understanding of the ionic environment role in protein-rRNA interactions is still rudimentary, and few exceptions have emerged regarding the role of monovalent and divalent ions (Batey and Williamson 1998, Agalarov et al. 2000, Xing and Draper 1995, Conn et al. 1999, Drygin and Zimmermann 2000).