Inhibition of chloramphenicol binding to Escherichia coli 70S ribosomes by 2'(3')-O-aminoacyl-dinucleoside phosphates derived from the aminoacyl-tRNA acceptor terminus.

The effect of 2' and 3'-O-aminoacyl-dinucleoside phosphates cytidylyl(3'-5')-2'(3')-O-L-phenyl-alanyladenosine (I), cytidylyl(3'-5')-3'-deoxy-2'-O-L-phenylalanyladenosine (IIa), cytidylyl(3'-5')-2'-deoxy-3'-O-L-phenylalanyladenosine (IIIa), cytidylyl(3'-5')-3'-deoxy-2'-O-glycyladenosine (IIb), cytidylyl(3'-5')-2'-deoxy-3'-O-glycyladenosine (IIIb), cytidylyl(3'-5')-3'-deoxy-2'-O-L-leucyladenosine (IIc), cytidylyl(3'-5')-2'-deoxy-3'-O-L-leucyladenosine (IIIc), cytidylyl(3'-5')-3'-O-L-phenylalanyladenosine (IIId) as analogs of the 2'(3')-aminoacyl-tRNA termini, on chloramphenicol binding to 70S Excherichia coli ribosomes was investigated. The association constants (Kb) of the investigated compounds were determined by the equilibrium dialysis method. Based on the constancy of Kb over the range of inhibitor concentration, it was determined that the binding site of the 2' isomers IIa-IIc overlaps with the chloramphenicol site, whereas the variability of Kb for the 3' isomers IIIb, IIIc and especially IIIa seems to indicate that they do not achieve a complete fit. The consistently higher values of the Kb values for the 3' isomers IIIa-IIIc relative to that of the 2' isomers IIa-IIc also indicate a stabilization of the binding of the former due to a specific interaction between its amino acid portion and a ribosomal site.     

Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond formation.     

Cooperative binding of 3'-fragments of transfer ribonucleic acid to the peptidyltransferase center of Escherichia coli ribosomes.

The binding of substrates to the A-site half (A′) and the P-site half (P′) of the peptidyltransferase center was measured by means of equilibrium dialysis. The tRNA fragments C-A-C-C-A-Leu and C-A-C-C-A-(N-acetyl)Leu were used as A′-site and P′-site substrates, respectively. The A′- and P′-substrates bound well to 50 S in contrast to 30 S subunits; significant binding to 23 S and 16 S RNA was also found. The binding of the P′-site substrate to 23 S RNA and 50 S subunits was very similar at various Mg²⁺ and K⁺ concentrations, indicating that the 23 S RNA is probably directly involved in the binding of the 3′-end of the peptidyl-tRNA. Cooperative effects at the peptidyltransferase center were found using chloramphenicol and deacylated tRNA as competitors, which completely inhibited the substrate binding to one site whilst drastically stimulating binding to the other. Chloramphenicol inhibited the binding of the A′-site substrate C-A-C-C-A-Leu, whereas the binding of the corresponding P′-site substrate was stimulated. In contrast, deacylated tRNA blocked the binding of the P′-site substrate, but stimulated the corresponding A′-site binding. Similarly, the trinucleotide C_p,C_pA inhibited binding of the P′-site substrate (showing complete inhibition at 70 μm) whereas binding of the A′-site substrate was slightly stimulated at concentrations below 70 μm.     

Distances between 3' ends of ribosomal ribonucleic acids reassembled into Escherichia coli ribosomes

The three ribonucleic acids (RNAs) from Escherichia coli ribosomes were isolated and then labeled at their 3' ends by oxidation with periodate followed by reaction with thiosemicarbazides of fluorescein or eosin. Ribosomal subunits reconstituted with the labeled RNAs were active for polyphenylalanine synthesis. The distances between the 3' ends of the RNAs in 70S ribosomes were estimated by nonradiative energy transfer from fluorescein to eosin. The percentage of energy transfer was calculated from the decrease in fluorescence lifetime of fluorescein in the quenched sample compared to the unquenched sample. Fluorescence lifetime was measured in real time by using a mode-locked laser for excitation and a high-speed electrostatic photomultiplier tube for detection of fluorescence. The distances between fluorophores attached to the 3' ends of 16S RNA and 5S RNA or 23S RNA were estimated to be about 55 and 71 A, respectively. The corresponding distance between the 5S RNA and 23S RNA was too large to be measured reliably with the available probes but was estimated to be greater than 65 A. Comparison of the quantum yields of the labeled RNAs free in solution and reconstituted into ribosomal subunits suggests that the 3' end of 16S RNA does not interact appreciably with other ribosomal components and may be in a relatively exposed position, whereas the 3' ends of the 5S RNA and 23S RNA may be buried in the 70S ribosomal subunit.     

Selective spin-labeling of the ribosomal proteins of 70S ribosomes from Escherichia coli.

We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1--2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 greater than S21, L27 greater than S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 A (1 A = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spinlabeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.     

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.     

Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes

To investigate ribosome topography and possible function, 70S ribosomes of $\text{Escherichia coli}$ were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.     

Non-enzymic binding of phenylalanyl transfer ribonucleic acid to ribosomes and ribosomal subunits from rat liver

• 1.1. (³H)phe-(³²P)tRNA was used to compare the binding of phe-tRNA to 80 S ribosomes and 40 S subunits from rat liver in circumstances where deacylation of bound tRNA could be measured.
• 2.2. About twice as much phe-tRNA was bound to the 80 S ribosome as to the 40 S subunit.
• 3.3. Deacylation of phe-tRNA bound to the 80 S ribosome did not occur but about 20 % of the phetRNA bound to the 40 S subunit was lost by deacylation.

     

Zn(II) binding to Escherichia coli 70S ribosomes

Escherichia coli 70S ribosomes tightly bind 8 equiv of Zn(II), and EXAFS spectra indicate that Zn(II) may be protein-bound. Ribosomes were incubated with EDTA and Zn(II), and after dialysis, the resulting ribosomes bound 5 and 11 equiv of Zn(II), respectively. EXAFS studies show that the additional Zn(II) in the zinc-supplemented ribosomes binds in part to the phosphate backbone of the ribosome. Lastly, in vitro translation studies demonstrate that EDTA-treated ribosomes do not synthesize an active Zn(II)-bound metalloenzyme, while the as-isolated ribosomes do. These studies demonstrate that the majority of intracellular Zn(II) resides in the ribosome.     

In vitro binding of 70S ribosomes to membrane structures of Escherichia coli

It was found that the maximal disattachment of the ribosomes from the membrane structures is observed upon their treatment with 10 mM tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 750 mM KCl, 5 mM magnesium acetate and 1 mM EDTA or puromycin. The most effective attachment of ribosomes to the membrane occurs in 10 mM tris-HCl buffer, pH 7.5, containing 5% sucrose and Mg2+. The increase of Mg2+ concentration in the medium from 0.5 mM up to 1 mM results in a 2-fold increase of the ribosomes bound to the membranes. The concentration of the ribosomal material involved in the reaction is very essential for ribosome binding to the membranes. The amount of ribosomes bound to the membranes increases proportionally to the increase of the ribosome concentration in the reaction mixture.     

Association of messenger ribonucleic acid with 70S monosomes from down-shifted Escherichia coli.

The complexed 70S ribosomes (monosomes) that accumulate in Escherichia coli after an energy source shift-down were examined in an electron microscope. In all cases, the ribosomes lie at or near one end of a ribonucleic acid (RNA) strand. This messenger RNA (mRNA) has a mean length of 168 nm and a length-average length of 200 nm, sufficient to code for polypeptides of a weight-average molecular weight of 20,000. The length distribution indicates that these strands are a reasonable representation of the population of monocistronic mRNA's of E. coli. The mRNA strands disappear entirely upon digestion with pancreatic ribonuclease, phosphodiesterase I, or polynucleotide phosphorylase. The susceptibility to digestion by 3'-exonucleases indicate that the ribosomes lie at the 5' end of the mRNA strands. These results are consistent with the hypothesis that down-shifted cells have a translational defect at a point subsequent to the binding of ribosomes to mRNA but prior to the formation of the first peptide bond, such that ribosomes remain bound at or near their points of initial attachment to mRNA.