Proflavine binding to yeast rRNA and ribosomes as related to structure

Proflavine binding experiments were carried out with yeast rRNA, native and “unfolded” ribosomes; the binding constants and the number of binding sites were calculated by a spectroscopic method. The study of the intercalation complexes by fluorescence and electric dichroism shows the intercalation binding sites to involve two subtypes of sites, which could be related to different nucleotide composition and secondary structure of the rRNA regions, i.e., binding sites located in the (A + U)-rich single strands and binding sites located in the (G + C)-rich double-helical strands (fluorescence quenching sites). Electric dichroism of complexed proflavine is interpreted in terms of rRNA conformation within the ribosomes. The conclusions are in agreement with the ribosomal model of Cox and Bonanou and show that, according to this model, the base planes of the nucleotides are not all parallel in the native ribsome, but rather radiate around the folding axis of the ribonucleoprotein sheet.     

Fluorescence studies of the binding of a yeast tRNAPhe derivative to Escherichia coli ribosomes.

The equilibrium binding of a highly fluorescent derivative of yeast tRNA^Phe to Escherichia coli 70 S ribosomes was studied fluorimetrically at 7 °C in 25 mm-magnesium. Under these conditions 70 S ribosomes bind two deacylated tRNAs stoichiometrically. An analysis of the binding data using a model in which occupancy of the weaker site requires prior occupancy of the stronger site leads to apparent association constants of (1.00 ± 0.05) × 10⁹m^?1 and (3.4 ± 0.2) × 10⁷m^?1. The use of an independent site model does not change these values appreciably. The observed binding constants do not depend upon the presence or absence of the messenger RNA, poly(U). However, spectroscopic evidence strongly suggests that the anticodons of both bound tRNAs are in contact with the message. This evidence further suggests that in the presence of poly(U) the environment of the hypermodified base adjacent to the anticodon is substantially different in the two sites. This may reflect a difference in the conformation of the anticodon loops or an interaction between the hypermodified base of the weak site tRNA and the anticodon loop of the strong site tRNA.     

Phenylalanyl-tRNA synthetases from sheep liver and yeast. Correlation between net charge and binding to ribosomes

Unlike phenylalanyl-tRNA synthetase from lower eukaryotes, the corresponding enzyme from higher eukaryotes displays a pronounced tendency to associate with ribosomes in vitro. To attempt to uncover the structural features responsible for this difference in behavior, a comparative study of the enzymes purified to homogeneity from sheep liver and yeast was undertaken. The two alpha 2 beta 2-type enzymes displayed remarkably similar subunit molecular masses (71 and 63 kDa for sheep, 74 and 63 kDa for yeast), yet differed markedly in their isoelectric points (8.0 and 5.6 pH units, respectively). Mild tryptic digestion of the enzyme from sheep led to preferential degradation of the 63-kDa beta subunit into two major fragments of 35 and 25 kDa, respectively, with concomitant loss of activity. The isoelectric points of the denatured fragments were found to be distinctly lower than that of the denatured beta subunit, implying that the residues responsible for the basic net charge of the original beta subunit are mainly clustered in a small portion of the polypeptide chain which was excised during proteolysis. Despite their different isoelectric points, the enzymes from yeast and sheep displayed identical requirements for aminoacylation of tRNA at optimal rates. Moreover, the incidence of variations in pH and ionic strength on the kinetic parameters of the two enzymes was indistinguishable. Interpreted in terms of the polyelectrolyte theory, these results support the view that the residues responsible for the basic net charge of the mammalian enzyme are located in a region distal from the active site. It is suggested that the cationic charge of the enzyme allows anchorage to a cellular component carrying negative charges, possibly the ribosome.     

The binding of kinetin to plant ribosomes

The synthetic cytokinins kinetin and 6-benzylaminopurine exhibit equilibrium-type binding to purified chinese-cabbage leaf ribosomes. At 23mum and 4 degrees C one molecule of kinetin and 1.34 molecules of 6-benzylaminopurine are bound per ribosome. Adenine and adenine derivatives that are inactive as cytokinins showed much less affinity for ribosomes. Pretreatment of ribosomes with 0.5m-ammonium chloride or Triton X-100 did not decrease the extent of cytokinin binding. Binding appeared to be to the 83S ribosome species. A positive correlation between the extent of binding and the biological effect of various cytokinin analogues was demonstrated. These results are discussed in terms of cytokinin control of growth processes at the ribosomal level.     

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

Adhesive binding of rokitamycin to Staphylococcus aureus ribosomes

Rokitamycin (RKM), a 3"-O-propionyl derivative of leucomycin A5, is bactericidal against staphylococci near the minimum inhibitory concentrations. RKM bound to ribosomes before-hand is only slightly displaced by erythromycin or josamycin, or even by RKM itself. The adhesive binding of the RKM-ribosome complex might prove to be the lethal event for susceptible staphylococci.     

RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes

Here, we examine tRNA-aminoacyl synthetase (ARS) localization in protein synthesis. Proteomics reveals that ten of the twenty cytosolic ARSs associate with ribosomes in sucrose gradients: phenylalanyl-RS (FRS), and the 9 ARSs that form the multi-ARS complex (MSC). Using the ribopuromycylation method (RPM) for localizing intracellular translation, we show that FRS and the MSC, and to a lesser extent other ARSs, localize to translating ribosomes, most strikingly when translation is restricted to poxvirus or alphavirus factories in infected cells. Immunoproximity fluorescence indicates close proximity between MSC and the ribosome. Stress induced-translational shutdown recruits the MSC to stress-granules, a depot for mRNA and translation components. MSC binding to mRNA provides a facile explanation for its delivery to translating ribosomes and stress granules. These findings, along with the abundance of the MSC (9 × 10(6) copies per cell, roughly equimolar with ribosomes), is consistent with the idea that MSC specificity, recently reported to vary with cellular stress (Netzer, N., Goodenbour, J. M., David, A., Dittmar, K. A., Jones, R. B., Schneider, J. R., Boone, D., Eves, E. M., Rosner, M. R., Gibbs, J. S., Embry, A., Dolan, B., Das, S., Hickman, H. D., Berglund, P., Bennink, J. R., Yewdell, J. W., and Pan, T. (2009) Nature 462, 522-526) can be modulated at the level of individual mRNAs to modify decoding of specific gene products.     

Mechanism of mRNA binding to bovine mitochondrial ribosomes

The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.     

Terbium binding to ribosomes and ribosomal RNA.

Terbium binding to rat liver ribosomes and ribosomal RNA (rRNA) was examined by equilibrium dialysis and fluorescence spectroscopy. Upon binding to ribosomes and rRNA, the enhancement of terbium fluorescence emission at both 488 and 541 nm was dependent only upon the amount of bound terbium and independent of ionic strength. Binding profiles for ribosomes and rRNA suggested that terbium was bound to ribosomes primarily through rRNA interactions. Data suggested that terbium mimicked characteristics previously described for interactions between ribosomes and magnesium. It is proposed, therefore, that fluorescence of terbium bound to ribosomes may prove useful in studies on the nature and extent of interactions between ribosomes and magnesium.