A modified method of isolation of "tight" 70S ribosomes from Escherichia coli highly active at different stages of the elongation cycle

The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80%. We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's. These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1. short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i. e. in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2. washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3. final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl. "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.     

Interaction of tRNA with ribosomes

Definition of the site of tRNA-binding to ribosomes is suggested on the basis of a free energy of tRNA-ribosome interaction. From this point of view disagreements that have arisen in recent years concerning the numbers of tRNA binding sites on the ribosome, their distribution between subunits, the properties of the third site E in ribosomes and the compatibility of new experimental data with different models of elongation cycle are discussed. The observation of the third site in the ribosome (messenger independent and with a presumably exit function) is not a refutation but an extension of Watson's model of translating ribosome.     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.     

Mechanism of tRNA Translocation on the Ribosome

During the translocation step of the elongation cycle of peptide synthesis two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. Translocation is catalyzed by the elongation factor G (EF-G) and requires GTP hydrolysis. The fundamental biochemical features of the process were worked out in the 1970–80s, to a large part by A.S. Spirin and his colleagues. Recent results from pre-steady-state kinetic analysis and cryoelectron microscopy suggest that translocation is a multistep dynamic process that entails large-scale structural rearrangements of both ribosome and EF-G. Kinetic and thermodynamic data, together with the structural information on the conformational changes in the ribosome and EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.     

Effect of adrenalectomy of the recipients of allogeneic lymphocytes on the inactivation of endogenous colony-forming cells in mice

Intravenous injection of lymph node cells from the parental C57BL/6 mouse line in doses of 2 X 10(6) or 5 X 10(6) into sublethally irradiated (CBA X C57BL/6) F1 hybrids produced more demonstrable suppression of endogenous colony-formation in adrenalectomized recipients as compared with that seen in sham-operated on ones (P less than 0.01). The recipients' adrenalectomy itself was accompanied by an over 2-fold increase in the number of the endogenous colony-forming cells in the spleen as compared with sham-operated on mice. Possible mechanisms, by which the killer action of lymphocytes on the endogenous colony-forming cells is potentiated, are under discussion.     

Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy

Poly(U) with an average chain length of 40-70 nucleotides was modified at the 5'- or 3'-terminal residues with 2,4-dinitrophenyl derivatives. The modified poly(U) was used to form 30S.poly(U) or 70S.poly(U).Phe-tRNA complexes. Localization of the 5' and 3' ends of the template polynucleotide on the 30S subunit and the 70S ribosome was performed by immune electron microscopy using antibodies against dinitrophenyl haptens. The 5' and 3' ends of poly(U) (putative entry and exit sites of the message) were found in the same region both on the 30S subunit and the 70S ribosome. They were located on the dorsal side of the 30S subunit between the head and the body near the groove bordering the side ledge (platform). Comparison of the size of this region with the possible length of the polynucleotide chain covered by the ribosome allowed us to suggest that the message makes a 'U-turn" (or forms a 'loop') as it passes through the ribosome.     

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.     

Number of tRNA binding sites on 80 S ribosomes and their subunits

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5-20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA.     

The Effect of Modification of Nucleotide-37 on the Interaction of Aminoacyl-tRNA with the A Site of the 70S Ribosome

To estimate the effect of modified nucleotide 37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNA^Phe _+Y and Phe-tRNA^Phe _–Y) with the A site of complex [70S · poly(U) · deacylated tRNA^Phe in the P site] was assayed at 0–20°C. As comparisons with native Phe-tRNA^Phe _+Y showed, removal of the Y base decreased the association constant of Phe-tRNA^Phe _–Y and the complex by an order of magnitude at every temperature tested, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA^Phe bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNA^Phe _–Y but not for Phe-tRNA^Phe _+Y. Thus, the modified nucleotide 3" of the Phe-tRNA^Phe anticodon stabilized the codon–anticodon interaction both in the A and P sites of the 70S ribosome.