The distance between two functionally significant regions of the 50 S Escherichia coli ribosome: the erythromycin binding site and proteins L7L12

The distance between the erythromycin binding site on the 50 S Escherichia coli ribosome and protein L7 has been measured by singlet-singlet energy transfer. A non-covalently bound erythromycin derivative, fluoroscein isothiocyanate erythromycylamine, was used as the acceptor. This derivative can be completely displaced from ribosomes by erythromycin, suggesting that they have the same binding site. 1,5-Iodoacetylethylenediamine naptholsulfonate-labeled protein L7 served as the fluorescent donor. It was reconstituted with salt/ethanol-washed 50 S cores. This readdition was accompanied by total recovery of elongation factor G-dependent GTPase activity. This suggests that the protein modification does not significantly perturb 50 S function or structure. Energy transfer measurements by both static and lifetime techniques were in good agreement. After consideration of various errors that enter the measurements and calculations, the L7-erythromycin distance is estimated to be 70 ± 10 Å. This long distance is interesting, since both sites may be involved in translocation.The fluorescent derivative of erythromycin was also used to study binding kinetics to the 50 S and 70 S ribosomes. Binding is a simple second-order step and proceeds about 11 times faster on the 70 S particle. Exchange of the fluorescent derivative with excess erythromycin is limited by the dissociation rate, and this is four times faster on the 70 S particle. These results suggest that the erythromycin site is more accessible on the 70 S particle, and may be an indication of conformational changes in the 50 S ribosome upon combination with the 30 S ribosome.     

Kinetic studies of the rates and mechanism of assembly of the protein synthesis initiation complex.

Rate constants for a number of the assembly reactions involved in forming Escherichia coli ribosome initiation complexes have been measured. These reactions were monitored in a stopped-flow device in which Rayleigh scattering and fluorescence anisotropy were followed as a function of time. Fluorescence was induced by laser excitation modulated at 50 kHz. Aminoacyl-tRNA, initiation factor 3 (IF3), and 70S ribosomes were labeled with fluorescent probes. The light-scattering and fluorescence data show that the antiassociation model for IF3 function cannot be correct. IF3 can be considered to act as an effector in an allosteric model for ribosome function. Fluorescence anisotropy stopped-flow experiments provided rate constants for the binding of IF3 to both 30S subunits and to the intact 70S ribosome. Aminoacyl-tRNA's and nucleotide triplets appear to bind rapidly to 70S ribosomes and then a slow first-order conformational change occurs.     

Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

Design, synthesis, and biological evaluation of BODIPY-erythromycin probes for bacterial ribosomes

BODIPY-erythromycin probes of bacterial ribosomes were designed and synthesized by attaching a BODIPY fluorophore to the 4'- and 9-positions of the erythromycin structure. The probes exhibited excellent binding affinity to bacterial ribosomes and competed with erythromycin and other drugs whose binding sites are in the same vicinity of the 50S subunit. The synthetic fluorescent probe 5 was successfully adapted in our ultra high-throughput screening (uHTS) to identify novel ribosome inhibitors.     

Localization of the elongation factor Tu binding site on Escherichia coli ribosomes

Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.     

A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.

70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

RNase catalyzed hydrolysis of ribosomes in several functional states

The RNase A catalyzed hydrolysis of rRNA in ribosomes has been studied for nonwashed 50S and 70S ribosomes, for washed 50S and 70S ribosomes, for runoff 50S ribosomes and for 70S ribosomes in polysomes. The regions available to hydrolysis in the 50S ribosome remain available when the 50S ribosomes become a part of a 70S ribosome or a polysome. The regions available to hydrolysis in the 30S ribosome become unavailable when the 30S ribosome becomes part of a 70S ribosome or a polysome. Removal of tRNA, mRNA and factors from the 50S and 70S ribosome lowers the rate of hydrolysis of one site in the 23S rRNA. This shows that the conformation of one region of the 23S RNA changes for ribosomes in different functional states.     

New fluorescent macrolide derivatives for studying interactions of antibiotics and their analogs with the ribosomal exit tunnel

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.     

Identification of a rRNA/chloramphenicol interaction site within the peptidyltransferase center of the 50 S subunit of the Escherichia coli ribosome.

We have used oligodeoxyribonucleotide probes to investigate possible interactions between chloramphenicol and portions of the rRNA contained within the peptidyltransferase center of the Escherichia coli ribosome. Oligodeoxyribonucleotide probes complementary to bases 2448-2454, 2468-2482, and 2497-2505 of 23 S rRNA were hybridized to 50 S subunits in situ. Probe binding was qualitatively assessed by sucrose gradient centrifugation. Each probe was shown to bind specifically with its intended binding site through digestion of the rRNA within the RNA/DNA hetero-duplexes with RNase H and analysis of the digestion fragments using gel electrophoresis. Competitive binding experiments were conducted between each probe and the antibiotics chloramphenicol and erythromycin. The binding of a probe complementary to bases 2497-2505 was attenuated by 70% upon the binding of chloramphenicol. A probe complementary to bases 2468-2482 showed an increase in binding of 14% while binding of a probe complementary to bases 2448-2454 was not affected by chloramphenicol binding. Erythromycin did not affect the binding of any of these probes to 50 S subunits. These results suggest that bases within the 2497-2505 region of 23 S rRNA in E. coli may be involved in a chloramphenicol/rRNA interaction.     

Characterisation of the binding of virginiamycin S to Escherichia coli ribosomes.

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit.     

Defective assembly of the mitochondrial ribosomes in yeast cells grown in the presence of mitochondrial protein synthesis inhibitors

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in the presence of mitochondrial protein synthesis inhibitors erythromycin and chloramphenicol. Yeast cells grown in the presence of erythromycin (2 mg/ml) do not appear to contain any detectable amounts of the mitochondrial small (37 S) ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S was observed; this particle could be shown to be related to the mitochondrial small ribosomal subunit by two-dimensional gel electrophoretic analysis of its protein components. Since the var1 protein is the only mitochondrial translation product known to be associated with the mitochondrial ribosome, our results suggest that this protein is essential for the assembly of the mature small subunit, and that the var1 protein enters the pathway for the assembly of the small subunit at a late step. In at least one strain of yeast the accumulation of the 30-S particle appears to be very sensitive to catabolite repression. When yeast cells are grown in the presence of chloramphenicol instead of erythromycin, assembly of the small subunit appears to be only partially inhibited, and the presence of the 30-S particle could not be clearly demonstrated. This observation is consistent with the fact that in yeast, chloramphenicol inhibits mitochondrial protein synthesis by about 95% only and that the synthesis of the var1 protein appears to be the least sensitive to this inhibition.     

Cooperative and antagonistic interactions of peptidyl-tRNA and antibiotics with bacterial ribosomes.

There is a single-site interaction of [methylene-14C]thiamphenicol and [methylene-14C]chloramphenicol with run-off ribosomes with dissociation constants Kd = 6.8 micronM and Kd = 4.6 micronM respectively. Similar affinities for the antibiotics are observed in polysomes totally deprived of nascent peptides, or bearing nascent peptides on the A-site. However, two types of interaction are observed in endogenous polysomes with some ribosomes bearing nascent peptides on the P-site and other in the A-site. The lower-affinity bindings (dissociation constants Kd = 6.4 micronM and Kd = 1.5 micronM for thiamphenicol and chloramphenicol respectively) are due to the ribosomes bearing nascent peptides on the A-site. The higher-affinity bindings (dissociation constants Kd = 2.3 micronM and Kd = 1.5 micronM for thiamphenicol and chloramphenicol, respectively) are due to the ribosomes bearing nascent peptides on the P-site. Therefore binding of nascent peptides to the A-site does not affect the affinities of thiamphenicol and chloramphenicol for the ribosome. On the other hand interaction of the nascent peptides with the P-site of the ribosomes increases the affinities of both antibiotics for the ribosome. Thiamphenicol and chloramphenicol are thus good inhibitors of peptide bond formation in ribosomes and polysomes. Their affinities are increased precisely when the peptidyl-tRNA is placed in the P-site preceeding the peptide bond formation step, which is specifically blocked by the antibiotics. There is a single-site interaction per ribosome for [35S]thiostrepton, which does not appear to be affected by the attachment to the ribosomes of mRNA, tRNA and nascent peptides either to the A or the P-site. [N-methyl-14C]Lincomycin, [N-methyl-14C]erythromycin, [G-3H]streptogramin B and [G-3H]-streptogramin A bind to run-off ribosomes and polysomes totally free from nascent peptides. However, these antibiotics do not interact with ribosomes bearing nascent peptides either in the A or the P-site and therefore are not active on preformed polysomes. Thus lincomycin and streptogramin A only interact with free ribosomes and 50-S subunits and block the early rounds of peptide bond formation prior to polysome formation. Erythromycin and streptogramin B do not inhibit either initiation or the first round of peptide bond formation. However, erythromycin and streptogramin B, prebound to the ribosome, block peptide elongation probably by steric hindrance with the growing oligopeptide chain when this reaches a certain critical length.     

Enhancement of peptidyl transferase activity by antibiotics acting on the 50 S ribosomal subunit

Interconversions of ribosomes, between forms that are active and inactive in peptidyl transfer, were studied and conditions favoring a state of equilibrium between the two forms were established. Under such conditions activity was enhanced two-to fivefold by the antibiotics erythromycin, vernamycin Bα, lincomycin, chloramphenicol and vernamycin A. The antibiotics puromycin, gougerotin, thiostrepton and siomycin, whose target site is also the 50 S ribosomal subunit, were ineffective.A common feature of the effective antibiotics is their ability to bind to ribosomes active in peptidyl transfer but not to enzymatically inactive ribosomes. The activity enhancing effect of antibiotics is therefore interpreted as being due to a shift in the equilibrium between the two ribosomal forms in favor of the active conformation, brought about by the preferential binding of the antibiotic to ribosomes in this form. The results stress the flexible nature of ribosome structure and suggest that antibiotics can function as allosteric effectors in modifying ribosome conformation.     

The extension of polyphenylalanine and polylysine peptides on Escherichia coli ribosomes

Fluorescence techniques were used to examine aminoacyl-tRNA binding to Escherichia coli ribosomes and the subsequent extension of polyphenylalanine and polylysine nascent peptides. The results demonstrate that deacylated tRNA, an analogue of peptidyl-tRNA and puromycin (an analogue of aminoacyl-tRNA) can be bound simultaneously to the same ribosome. Moreover, the fluorescence properties of nascent polyphenylalanine and polylysine peptides with a fluorophore attached to their amino termini were determined and found to be quite different. This difference is reflected in the effects that erythromycin has in each case.     

Streptomycin-induced formation of 70-S monosomes and oligomers in Chlamydomonas reinhardi

Under ionic conditions, where the 70 S ribosomes but not the 80 S ribosomes partly dissociate into the subunits, in three mutants of Chlamydomonas reinhardi streptomycin causes in vivo at first an increase, later a decrease of the 70 S ribosome fraction. This behaviour can be explained, if streptomycin acts on the ribosome cycle of the organelle ribosomes of eukaryotes in the same way as on the ribosome cycle of E. coli.Streptomycin also induces the formation of dimers and oligomers from 80 S cytoplasmic ribosomes. The kinetics of this formation is similar to that of the 70 S ribosomes. However, this effect of streptomycin does not seem to influence the functional capacity of the 80 S ribosomes.     

Ribosome binding by tRNAs with fluorescent labeled 3'' termini.

Yeast and E. coli tRNAPhe samples were oxidized and labeled at the 3' end with dansyl hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmed E. coli 70S tight couple ribosomes in 25 mM magnesium at 8 degrees C. Two binding sites with binding constants of about 1 X 10(9) M-1 (P) and 3 X 10(7) M-1 (A) were determined for the yeast tRNAPhe derivatives. With E. coli tRNAPhe the A site affinity is similar to yeast tRNAPhe but the P site affinity is 5-fold weaker. Singlet-singlet energy transfer showd that the distance from the 3' end of tRNAPhe in the P site to a fluorescein derivative of erythromycin is 23 A. This supports in vitro studies suggesting that erythromycin binds near the peptide moiety of peptidyl tRNA. A distance of 34 A between the 3' ends of 2 tRNAs bound simulatneously on the ribosome was also measured. This long distance may mean that the deacylated fluorescent tRNA binds to the A site in an orientation like that in the stringent response rather than in protein synthesis.     

Studies on the binding of N-bromoacetylpuromycin and N-bromoacetylaminonucleoside to rat liver ribosomes.

[3H]N-Bromoacetylaminonucleoside and [3H]N-bromoacetylpuromycin have been synthesised as possible alkylating agents in order to study their interactions with rat liver ribosomes. Both compounds bind covalently to ribosomes to a considerable extent. The puromycin derivative binds to the extent of approximately 8 mol per ribosome, while the aminonucleoside derivative binds to the extent of approximately 13 mol per ribosome. Ammonium sulphate precipitation of ribosomes or treatment with puromycin, followed by washing of the ribosomes through NH4Cl-containing sucrose density gradients decreases the binding of both derivatives. Partial unfolding or denaturation of ribosomes by heating at 65 degrees C or through the action of various chemical reagents appears to expose more sites for binding. However, at 15 min of heating the binding of the puromycin derivative decreased by approximately 50% while the binding of the aminonucleoside derivative was almost zero. Binding of both labelled derivatives occurred only with the 50S ribosomal subunit. The extent of binding to the smaller 30S subunit was approximately 4% of that of the 50S subunit. Various other experiments are also described dealing with the binding of [3H]N-acetylphenylalanyl-tRNA to the A site of ribosomes following treatment with the N-bromoacetyl derivatives.     

Spatial proximity of the ribosomal mRNA binding center to the 50S subunit]

4-(N-2-chloroethyl-N-methylamino)benzylamide of 5'-heptaadenylic acid was used for affinity labelling of the ribosome in the vicinity of its mRNA-binding centre. This derivative, similar to the free oligonucleotide, stimulates the binding of [14C]-lysyl-tRNA to ribosomes of E. coli and alkylates ribosomes both the 30S and the 50S subunits. The alkylation of ribosomes is inhibited by pre-incubation of ribosomes with polyadenylic acid, which suggests that the chemical modification is a specific one and occurs in the vicinity of mRNA-binding site. The fact, that a short oligonucleotide having an active group on its 5'end attacks the 50S subunit of ribosome may indicate that the mRNA-binding centre is located in the contact region between ribosomal subunits.     

Effect of Sulfhydryl Reagents on the Ribosomes of Bacillus subtilis

The effect of various sulfhydryl reagents on the ribosomes of Bacillus subtilis was studied. The 70S ribosomes were completely dissociated into 30S and 50S subunits by appropriate concentrations of p-chloromercuribenzoic acid (PCMB) and 5,5'-dithio-bis-(2-nitro-benzoic acid). The N-ethylmaleimide and iodoacetamide failed to dissociate the ribosomes even at relatively high concentrations. The rate of dissociation of ribosomes by PCMB varied with the concentration of ribosomes. A progressive decrease in the rate of dissociation was observed as the concentration of ribosomes in the reaction mixture was increased. The PCMB-induced ribosomal subunits were unable to reassociate into 70S monomers unless they were dialyzed against buffer containing beta-mercaptoethanol. On the average, four molecules of PCMB per 70S ribosome and two molecules of PCMB per each 30S and 50S subunit were bound. The number of PCMB molecules bound per ribosome did not change with increasing concentrations of PCMB, even though higher concentrations of PCMB resulted in dissociation of ribosomes into subunits.