Ribosomes magnesium ions

R. H., Draper, D. E. The RNA-binding domain of ribosomal protein Lll recognizes an rRNA tertiary structure stabilized by both fhiostrepton and magnesium ion. Nucleic Adds Res 2000, 28, 1778—... 

The glycoside/aminoglycoside antibiotics, like the macrolides, exert a bacteriostatic effect due to selective inhibition of bacterial protein synthesis, with the exception of novobiocin (26). The compounds neomycin (27), spectinomycin (28) and streptomycin (29) bind selectively to the smaller bacterial 30S ribosomal subunit, whilst lincomycin (30) binds to the larger 50S ribosomal subunit (cf. macrolides). Apramycin (31) has ribosomal binding properties, but the exact site is uncertain (B-81MI10802). Novobiocin (26) can inhibit nucleic acid synthesis, and also complexes magnesium ion, which is essential for cell wall stability. 

The uptake of Mn2+ or Co2+ by system I is inhibitory or lethal respectively, and leads to efflux of Mg2+ from the cell. It is likely that accumulated Mn3+ or Co2+ displaces Mg2+ from ribosomes, thus increasing intracellular free magnesium ions, which leave the cell via the Mg2+ transport systems. 

EF-Tu is a GTPase that positions aminoacyl-tRNA complexes within the A site of prokaryotic ribosomes during protein synthesis. The binding of a GTP molecule (red) by EF-Tu causes domain conformation changes (not shown) that result in the creation of a binding cleft for an aminoacyl-tRNA complex. GTP hydrolysis causes domains 1 and 2 to move apart so that the aminoacyl-tRNA (not shown) is released. (A magnesium ion is illusttated with a yellow sphere.)... 

Now let us consider the case of essential metal Ions that, nevertheless, produce deleterious effects in the wrong concentration. Magnesium Ions are required for protein synthesis, yet Mg " " Ions in too high concentration lead to errors, as is illustrated by the studies of Szer and Ochoa (16) on the incorporation of phenylalanine and leucine in a ribosomal preparation using poly(U) as the messenger RNA. UUC as well as the UUU codon in poly(U) code for phenylalanine, so that incorporation of the latter represents correct translation. UUA and UUG code for leucine, so that leucine incorporation in this system is "incorrect." At low Mg2+ concentration only phenylalanine is in fact incorporated. Phenylalanine incorporation is maximal at lOmM Mg " " as the Mg " " concentration is increased, however, leucine also becomes incorporated and its maximal incorporation is at 20mM Mg2+ ( ). 

Photoaffinity Reaction. Ribosomes, 70 S ( vacant couples, which do not dissociate at 6 roM magnesium ion concentration) are obtained from E. coli, strain MRE 600, according to the procedure of Noll et Incubation and irradiation are carried out in volumes of 0.5-1 ml. In a total volume of 1 ml are contained the following E. coli ribosomes (1.0 mg, 0.38 nmole) 3-(4-benzoylphenyl)propionyl-[ H]Phe-tRNA (0.01 mg, 0.4 nmole) or 3-benzoylpropionyl-[ H]Phe-tRNA (0.01 mg, 0.4 nmole) 0.2 mg of polyuridylic acid (Boehringer, Mannheim) dissolved in 50 mM NH4CI 50 mM HEPES (N-2-hydroxyethylpiperazine-iV -2-ethanesulfonic acid) sodium salt (pH 7.4) 10.5 mM magnesium acetate 0.5 mM EDTA and 6 mM mercaptoethanol. Samples are mixed at 0° and incubated for 15 min at 37°. When sensitivity to puromycin is tested, the drug is added to a concentration of 1 mM and the incubation is continued for an additional 15 min at 37°. 

Actinomycin D is remarkably specific in inhibiting the synthesis of ribosomal RNA (DNA-primed) without any effect on the synthesis of DNA (Reich et al,y 1962). Use of radioactive actinomycin showed that it was covalently bound to the guanine of the DNA starter, and did not combine with any other cell component at concentrations that block RNA synthesis. The phenoxazine nucleus intercalates into DNA near to a G-C pair, and the peptide portions project into the minor groove (Muller and Crothers, 1968) (for more on intercalation see Section 10.3b). Whereas the action of the aminoacridines is sensitive to magnesium ions and insensitive to urea, precisely the reverse is true of actinomycin. Again, proflavine prevents the enzymatic synthesis of guanine-free polynycleotides, but actinomycin does not. 

The documentation, by example, of ail of the major Mg-requiring enzymes of the body would lead to a very long discussion. Magnesium is required at various steps in the synthesis of DNA, RNA, and protein. The metal ion is required by various enzyn es participating in the synthesis of carbohydrates and lipids. Mg ions play a structural role, where they are bound to phospholipid membranes, ribosomes, and chromatin. Mg Lons are required by enzymes that are used in transmitting signals w ithin cells. These enzymes include adenylate cyclase, w hich... 

Experiments show that it is on the ribosomes that protein synthesis actually occurs. If cells synthesizing protein from radioactive amino acids are studied, the radioactivity is first found bound to the ribosomes, and is only later released from them as soluble protein. A cell suspension from which ribosomes have been removed can never be made to synthesize protein, whilst if they are subsequently replaced synthesis can proceed rapidly. But the ribosomes alone are inadequate. In order to incorporate radioactive amino acids into new protein there needs to be added to the ribosomes a preparation of soluble cell material which contains certain enzymes, the soluble low-molecular-weight messenger RNA (m-RNA), transfer RNA (t-RNA), ATP, GTP, and ions like magnesium and potassium. So what role do these various substances perform in protein synthesis ... 

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