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.     

Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of S21 to the 50S subunit and the effect of the 50S subunit on nonradiative energy transfer between the 3' end of 16S RNA and S21

Escherichia coli ribosomal protein S21 was labeled at its single cysteine group with a fluorescent probe. Labeled S21 showed full activity in supporting MS2 RNA-dependent binding of formylmethionyl-tRNAf to 30S ribosomal subunits. Fluorescence anisotropy measurements and direct analysis on glycerol gradients demonstrate conclusively that labeled S21 binds to 50S ribosomal subunits as well as to 30S and 70S particles. The relative binding affinities are in the order 70S greater than 30S greater than 50S. Other results presented appear to indicate that S21 is bound in the same position on either 50S subunits or 30S subunits as in 70S ribosomes, suggesting that the protein is bound simultaneously to both subunits in the latter. Addition of 50S subunits to 30S particles containing probes on S21 and at the 3' end of 16S RNA caused a decrease in the energy transfer between these points. The results correspond to an apparent change in distance from 51 to 61 A.     

Two monoclonal antibodies against Escherichia coli ribosomal protein L2 distinguish different locations for their respective epitopes in intact ribosomes

Two monoclonal antibodies raised against intact Escherichia coli ribosomal protein L2 were isolated, affinity-purified, and characterized. One of the antibodies (Ab 5-186) recognizes an epitope within residues 5-186, and the other (Ab 187-272) recognizes an epitope within residues 182-272. Both antibodies strongly inhibit in vitro polyphenylalanine synthesis when they are first allowed to bind to 50 S subunits prior addition of 30 S subunits. However, only Ab 187-272 is inhibitory when added to preformed 70 S ribosomes. Ab 5-186 binds to 50 S subunits but not to 70 S ribosomes. Ab 187-272 does not cause dissociation of 70 S ribosomes under the ionic conditions of the assay for polyphenylalanine synthesis (15 mM magnesium), although at 10 mM magnesium it does cause dissociation. Both antibodies inhibit the reassociation of 50 S with 30 S subunits. Both antibodies strongly inhibit peptidyltransferase activity. The two antibodies differ in their effects on interactions with elongation factors Tu (EF-Tu) and G (EF-G). Neither antibody significantly inhibits EF-G-dependent GTPase activity, nor the binding of EF-G when the antibodies are incubated with 50 S subunits; however, Ab 187-272 causes a decrease in the binding of EF-Tu X aminoacyl-tRNA X GTP ternary complex and of EF-Tu-dependent GTPase when it is incubated with 70 S ribosomes. The Fab fragments of both antibodies had effects similar to the intact antibodies. The results show that monoclonal antibodies can be used to discriminate different regions of L2 and that EF-Tu and EF-G do not have identical ribosomal binding sites.     

Aminoacyl-tRNA-elongation factor Tu-ribosome interaction leading to hydrolysis of guanosine 5'-triphosphate

We investigated the elongation factor Tu (EF-Tu) dependent binding of Phe-tRNA and Phe-tRNAs with the nicks at positions 46, 37, and 17 to the Escherichia coli 70S ribosome-poly(U)-tRNAPhe complex. Binding of Phe-tRNA1-45 + 47-76, Phe-tRNA1-36 + 38-76, or Phe-tRNA1-16 + 17-76 to the 70S ribosome has been found to be poly(U) X tRNA dependent and, similar to that of intact Phe-tRNA, is inhibited by the antibiotic thiostrepton. We have further found that, contrary to a previous report [Modolell, J., Cabrer, B., Parmeggiani, A., & Vazquez, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1796], the EF-Tu-ribosome GTPase mediated by Phe-tRNA is not inhibited by thiostrepton; rather, the drug stimulates the endogenous GTPase of the EF-Tu X 70S ribosome. Phe-tRNA fragments 47-76, 38-76, and 17-76 all promote the EF-Tu X GTPase reaction in the presence of 70S ribosome-poly(U)-tRNAPhe yeast. Moreover, since the GTPase-promoting activities of both the short and long fragments are similar, it appears that the most important aminoacyl transfer ribonucleic acid (aa-tRNA) interaction with EF-Tu occurs alongside its 3' quarter. Thiostrepton slightly stimulates the GTPase activity of these Phe-tRNA fragments. Although the Phe-tRNA1-36 + 38-76 cannot bind to poly(U) during its binding to 70S ribosomes, its binding at high Mg2+ concentration occurs at the A site. Thus, most of the bound modified Phe-tRNA functions as the acceptor in the peptidyltransferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The binding of ribosomal subunits to endoplasmic reticulum membranes

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60-80% of added 60S subunits and 10-15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane-60S subunit-40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.     

Comparison of ribosomal entry and acceptor transfer ribonucleic acid binding sites on Escherichia coli 70S ribosomes. Fluorescence energy transfer measurements from Phe-tRNAPhe to the 3' end of 16S ribonucleic acid

Distances were measured by nonradiative energy transfer from fluorescent probes specifically located on one of three points of yeast or Escherichia coli Phe-tRNAPhe enzymatically bound to the entry site or to the acceptor site of E. coli 70S ribosomes to energy-accepting probes on the 3' end of the 16S ribonucleic acid (RNA) of the 30S subunit. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin. Fluorescein isothiocyanate was attached to the X base (position 47) of E. coli tRNAPhe. E. coli tRNAPhe which had been photochemically cross-linked between positions 8 and 13 followed by chemical reduction to form a fluorescent probe was also used. Labeled tRNAs were aminoacylated and enzymatically bound to the ribosome in the presence of elongation factor Tu and guanosine 5'-triphosphate (acceptor-site binding) or a nonhydrolyzable analogue (entry-site binding). Nonradiative energy transfer measurements were made of the distances between fluorophores located on the Phe-tRNA and the fluorophore at the 3' end of 16S RNA. Calculations were based on comparison of the fluorescence lifetime of the energy donor, located on the Phe-tRNA, in the absence and presence of an energy acceptor on the 3' end of the 16S RNA. Under both sets of binding conditions, the distances to the 3' end of 16S RNA were found to be the following: cross-linked tRNA, greater than 69 A; Y base of tRNA, greater than 61 A. The distance between the 3' end of 16S RNA and the X base of tRNA was found to be 81 A under acceptor-site binding conditions but greater than 86 A under entry-site binding conditions.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.     

Functional interactions of an Escherichia coli ribosomal ATPase.

The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.     

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.     

Binding of spectinomycin by ribosomes fromEscherichia coli

The binding of the aminocyclitol antibiotic spectinomycin to 70S ribosomes and to 30S subunits fromEscherichia coli has been investigated. The association was influenced by the presence of messenger RNA. The K_d for [³H]-4 OH-spectinomycin binding to 70S ribosomes was 2×10^–7 M without mRNA (polyinosinic acid), and 1×10^–6 M with polyinosinic acid. Dissociation of the antibiotic from the ribosomes was significantly affected by the presence of a bound messenger RNA, which reduced the rate of dissociation by a factor of 5.7. The presence of mRNA did not influence the association of spectinomycin with the 30S subunit. The dissociation rate from the small subunit was comparable to the rate of dissociation from the 70S ribosome and was not affected by the presence of mRNA.     

Exploration of RNA 3' terminal locations in rat ribosome.

The locations of the 3' ends of RNAs in rat ribosome were studied by a fluorescence-labeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3' ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe - fluorescein 5-thiosemicarbazide (FTSC), indicating that the 3' termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3' terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3' end of 5 S RNA was located on the interface of two subunits. However, no fluorescence-labeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3' end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3' ends of ribosomal RNAs including oxidation with NaIO4, reduction with KBH4 and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3' ends of RNAs were not involved in the translation activity of ribosome.     

Area of 16S ribonucleic acid at or near the interface between 30S and 50S ribosomes of Escherichia coli.

To determine the region of 16S ribonucleic acid (RNA) at the interface between 30 and 50S ribosomes of Escherichia coli, 30 and 70S ribosomes were treated with T1 ribonuclease (RNase). The accessibility of 16S RNA in the 5' half of the molecule is the same in 30 and 70S ribosomes. The interaction with 50S ribosomes decreases the sensitivity to T1 RNase of an area in the middle of 16S RNA. A large area near the 3' end of 16S RNA is completely protected in 70S ribosomes. The RNA near the 3' end of the molecule and an area of RNA in the middle of the molecule appear to be at the interface between 30 and 50S ribosomes. One site in 16S RNA, 13 to 15 nucleotides from the 3' end, normally inaccessible to T1 RNase in 30S ribosomes, becomes accessible to T1 RNase in 70S ribosomes. This indicates a conformational change at the 3' end of 16S RNA when 30S ribosomes are associated with 50S ribosomes.     

Basepairing of oligonucleotides to the 3'' end of 16S ribosomal RNA is not stabilized by ribosomal proteins

We have studied the binding of the octanucleotide (5'-3')d(AAGGAGGT) which is fully complementary to the 3' end of 16S ribosomal RNA, to ribosomes and to the isolated target sequence (5'-3') (ACCUCCUUA). The binding constant for 30S or 70S ribosomes is (5 +/- 2) X 10(7) mol-1, whereas the duplex containing the octa- and the nonanucleotide has an association constant of (6 +/- 3) X 10(7) mol-1. The two values are the same within the experimental error. This result suggests that basepairing at the 3' end of 16S rRNA is not stabilized by ribosomal proteins.     

Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function.

The reaction pattern with N-[¹⁴C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg²⁺ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg²⁺-dependent. The reaction at 10 mm-Mg²⁺ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg²⁺; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg²⁺ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg²⁺. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg²⁺ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.     

Comparison of the reactions of chemically reactive analogs of U-G-A and of A-U-G with ribosomes of Escherichia coli.

The chemically reactive analog of U-G-A, 5'-(4-(Bromo-[2-14C] acetamido) phenylphospho) - uridylyl-(3'-5') - guanylyl-(3'-5') adenosine has a 20 fold lower affinity to 70S ribosomes than the corresponding analog of A-U-G though the U-G-A analog also preferentially reacts with protein S18 of 70S ribosomes. This reaction programs ribosomes for EF-T dependent Trp-tRNATrp-suIII binding. Therefore, it is concluded that this protein is part of the A'-site of the ribosomal codon binding site. Reaction of the U-G-A analog with 30S subunits lead to a predominant crosslinking of U-G-A to proteins S4 and S18. In contrast, a comparable reaction of the A-U-G analog with 30S subunits lead to a predominant crosslinking of A-U-G to proteins S4 and S12 (Pongs, O., Stoffler, G.A., Lanka, E., (1975) J. Mol. Biol. 99, 301). Since protein S12 is located at the 'P' site of the ribosomal codon binding site, it is proposed that the U-G-A analog does not bind at this site.     

The effects of initiation factor 3 on the formation of 30S initiation complexes with synthetic and natural messengers

Initiation factor IF-3 is required for the binding of fMet-tRNA to 70S ribosomes directed by AUG, poly (U,G), f₂RNA and T₄ late RNA as well as for the binding of acPhe-tRNA directed by poly (U). In contrast, IF-3 is not required for the binding of the initiator aminoacyl-tRNAs to isolated 30S subunits directed by the synthetic messengers, but is required for maximal formation of initiation complexes with natural messengers. These data indicate that with synthetic messengers the sole function of IF-3 is to dissociate the 70S ribosomes into subunits, whereas with natural messengers IF-3 is required not only for dissociation of the ribosomes but also for the binding of the messenger to the 30S subunit.