tRNA binding sites on the subunits of Escherichia coli ribosomes

Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity. Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA. Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent. The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies. Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site). Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes. In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA. Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site. The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes. 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits. The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.     

Chemical inactivation of Escherichia coli 30-S ribosomes by iodination. Identification of proteins involved in tRNA binding.

30-S ribosomal subunits are inactivated by iodination for both enzymic fMet-tRNA and non-enzymic Phe-tRNA binding activities. This inactivation is due to modification of the protein moiety of the ribosome. Reconstitutions were performed with 16-S RNA and mixtures of total protein isolated from modified subunits and purified proteins isolated from unmodified subunits. This allowed identification of the individual proteins which restore tRNA binding activity. S3, S14 and S19 were identified as proteins involved in fMet-tRNA binding. S1, S2, S3, S14 and S19 were identified as proteins involved in Phe-tRNA binding. Modified particles shown normal sedimentation constants and complete protein compositions both before and after reconstitution. This suggests that the loss of activity is due to modification of one or more of the actual binding sites located on the 30-S subunit and that restoration of activity is due to structural correction at this site rather than to correction of an assembly defect.     

Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes. Role of 50 S subunits in formation of the E site

The 30 S subunit contains 2 sites for tRNA binding (Phe-tRNA, AcPhe-tRNA, tRNAPheOH) with the functional properties of D and A sites of the 70 S ribosome after attachment of 50 S subunit. The third (E) site specific for deacylated tRNA is introduced into 70 S ribosome by its 50 S subunit. The E-site binding of tRNAPheOH is not sensitive to either tetracycline and edeine, and practically codon-independent. The affinity constant of tRNAPheOH for the E site is 2-3 orders of magnitude lower than that for the D site.     

Specific association of two homologous DNA-binding proteins to the native 30-S ribosomal subunits of Escherichia coli.

The native 30-S ribosomal subunits from Escherichia coli are shown to be associated with two proteins which are different from the known ribosome-associated and ribosomal proteins. Neither protein is foune on native 50-S subunits or on intact ribosomes in the cell extract. The purified proteins re-bind in vitro to free 30-S subunits, but do not bind to either free 50-S subunits or intact ribosomes. The proteins, denoted NS1 and NS2, have been purified and characterized. Both proteins showed the same molecular weight of 9500 by sodium dodecyl sulfate gel electrophoresis but 34 000 by gel filtration. Upon treatment with cross-linking reagents the purified proteins gave higher molecular weight species up to the tetrameric ones showing that they exist in solution as tetramers. The amino acid compositions, tryptic fingerprint patterns and N-terminal sequences of the two proteins have been determined. These data show that NS1 and NS2 possess distinct primary structures but with extensive sequence homology. Antibodies raised against the purified proteins cross-reacted in double immuno-diffusion tests confirming further the homology. Because of the similarity in properties a sample of the DNA-binding protein HD (Berthold, V. and Geider, K. (1976) Eur. J. Biochem. 71, 443--449) was compared to NS1 and NS2. In terms of several criteria, the protein HD is found to be a mixture of two proteins, namely NS1 and NS2. The present report is the first instance of an association of DNA-binding proteins to the ribosome.     

70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [3H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [3H]tRNA, or with [3H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the 'additional' site is not sensitive to the template. [3H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is preferred to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3'-end adenosine is essential for the affinity. The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli.

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5'-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9 S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3' end of the 16-S RNA, but lacking the 3'-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3'-terminal 50 nucleotides) was not found. This result suggests that the 3' region of 16-S RNA is not involved in stable interactions with protein.     

Comparative study of the interaction of polyuridylic acid with 30S subunits and 70S ribosomes of Escherichia coli.

Fractionated polyuridylic acid with an average chain length of 55 nucleotides forms binary complexes with 30S subunits with a stoichiometry of I:I. These complexes are heterogeneous in stability. The more stable one is characterized by an association constant K2 - 5.5xI09 M-I, and the less stable-by KI = I06xM-I, at 20 mM Mg2+, 200 mM NH4(+) and 0 degrees C. The main reason for this heterogeneity is the presence or absence of the ribosomal protein SI in the presence or absence of the ribosomal protein SI in the subunits. Decrease of Mg2+ concentration down to 5 mM hardly changes the K2 values but reduction of the NH4(+) concentration to 50 mM results in a 25-fold increase of K2. Association constants K2 for the stable complex, i.e. in the presence of SI protein, were measured at different temperatures (0 - 30 degrees C) and the thermodynamic parameters of binding (delta H degrees, delta S degrees, delta G degrees) were determined. Analogous experiments were made with 70S ribosomes. K2 values as well as delta H degrees, delta S degrees, delta G degrees appeared the same both for 30S and 70S ribosomes in all conditions examined. This is strong evidence that the 50S subunits do not contribute to the interaction of poly(U) with the complete 70S ribosomes.     

Leaderless mRNAs bind 70S ribosomes more strongly than 30S ribosomal subunits in Escherichia coli

By primer extension inhibition assays, 70S ribosomes bound with higher affinity, or stability, than did 30S subunits to leaderless mRNAs containing AUG or GUG start codons. Addition of translation initiation factors affected ribosome binding to leaderless mRNAs. Our results suggest that translation of leaderless mRNAs might initiate through a pathway involving 70S ribosomes or 30S subunits lacking IF3.     

Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two codon-specific binding sites for transfer RNA.

30S subunits were isolated capable to bind simultaneously two molecules of Phe-tRNAPhe (or N-Acetyl-Phe-tRNAPhe), both poly(U) dependent. The site with higher affinity to tRNA was identified as P site. tRNA binding to this site was not inhibited by low concentrations of tetracycline (2 x 10(-5)M) and, on the other hand, N-Acetyl-Phe-tRNAPhe, initially prebound to the 30S.poly(U) complex in the presence of tetracycline, reacted with puromycin quantitatively after addition of 50S subunits. The site with lower affinity to tRNA revealed features of the A site: tetracycline fully inhibited the binding of both Phe-tRNAPhe and N-Acetyl-Phe-tRNAPhe. Binding of two molecules of Phe-tRNAPhe to the 30S.poly(U) complex followed by the addition of 50S subunits resulted in the formation of (Phe)2-tRNAPhe in 75-90% of the reassociated 70S ribosomes. These results prove that isolated 30S subunits contain two physically distinct centers for the binding of specific aminoacyl- (or peptidyl-) tRNA. Addition of 50S subunits results in the formation of whole 70S ribosomes with usual donor and acceptor sites.     

Affinities of tRNA binding sites of ribosomes from Escherichia coli

The binding affinities of tRNAPhe, Phe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E. coli 70S ribosomes were determined at various ionic conditions. For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used. Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites. The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 10(8) M-1 and 5 X 10(9) M-1, respectively. Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration. Phe-tRNAPhe binds to the A site with a similar affinity. Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 10(4). Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore. The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site. In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength. Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficient codon-anticodon interaction in the E site. On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site. Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation.     

Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study

The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1). The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex. The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.     

The effect of modification of cytosines in Escherichia coli 16-S rRNA on reconstitution and function of 30-S ribosomes.

O-Methylhydroxylamine (methoxyamine) was used for selective modification of cytosine residues in Escherichia coli 16-S rRNA. It was shown that cytosines accessible for methoxyamination are randomly distributed along the 16-S rRNA chain. Preparations of methoxyaminated 16-S rRNA, containing 2--130 modified cytosines/chain, still retained the ability to bind 30-S proteins, but the physical assembly of reconstituted particles was incorrect. The protein compositions of the reconstituted and native particles did not differ qualitatively from each other. However, the amount of protein in reconstituted particles decreased with an increasing number of methoxyaminated cytosines in 16-S rRNA. The particles obtained sedimented slower than native 30-S subunits, lost their ability to associate with 50-S ribosomes and to bind native phage f2 RNA. In contrast, modification of 16-S rRNA did not affect binding of poly(U) by reconstituted particles.