Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers.

Interactions between Escherichia coli 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to cDNA oligomers, regions 517-528, 1397-1404, and 1534-1542. The binding of cDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNAPhe. Only the binding of cDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAPhe or tRNAfMet alone. The poly(AGU)/tRNAfMet ligand pair caused an additional decline in the binding of cDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the cDNA oligomers 517-528 or 1398-1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for cDNA 1534-1541 in the presence of mRNAs or tRNA. At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other ligands, caused a large decrease in the binding of the cDNA oligomers. The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight-couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [1].     

In vitro inactivation of ascites ribosomes by colicin E 3

Colicin E 3 treatment of 80 S ribosomes from mouse ascites cells completely arrests in vitro protein synthesis. Isolated 40 S subunits are resistant to the colicin action while the larger subunit becomes inactivated after treatment with this protein. 40 S subunits derived from colicin E 3 treated 80 S ribosomes lose their ability to participate in polyphenylalanine synthesis. Colicin E 3 damaged 80 S ribosomes appear to be functional with regard to Met-tRNA_f^Met binding while they fail to attach Phe-tRNA to the A-site. Thus, except for the susceptibility of their larger subunits to colicin, the inactivation mechanism of 80 S particles resembles the process which alters the bacterial ribosome.     

Mechanism of export of colicin E1 and colicin E3.

The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.     

An initiation factor causing dissociation of E. coli ribosomes

Purification of crude initiation factors, essential for polypeptide synthesis in cell-free systems of E. coli, yielded a fraction DF which causes dissociation of 70 S ribosomes. Its stoichiometric action on 70 S ribosomes is antagonized by increasing Mg(2+) concentrations but not by the addition of 30 S and 50 S subunits washed with high salt concentration. GTP did not stimulate this dissociating action. 2 &mgr;g of our most purified preparation caused 100% dissociation of 100 &mgr;g of 70 S ribosomes without added GTP. DF-induced dissociation is a very rapid process at 37 degrees C and is temperature-dependent in the range of 0 degrees -37 degrees C. DF, which is thermolabile factor, is much less or not effective with complexed 70 S ribosomes bearing peptidyl-tRNA and mRNA.     

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.     

Crystallization and preliminary x-ray investigation of colicin E3 in complex with its immunity protein

Crystals of the colicin E3-immunity protein complex have been grown from solutions of citrate at pH 5.6. The crystals are monoclinic, space group P2(1), with unit cell dimensions a = 67.71, b = 196.67, c = 85.58 A, and beta = 113.67 degrees. The crystals diffract to 3-A resolution and are stable in the x-ray beam for at least a day. Although the stoichiometry of the complex in solution is 1:1 there are two, three, or four such binary complex molecules in the asymmetric unit.     

On the interaction of colicin E3 with the ribosome

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

Inhibition of the initiation of leukemic transcription by N-trifluoroacetyladriamycin-14-O-hemiadipate in vitro. Impaired formation of RNA polymerase-DNA complex

The effect of N-trifluoroacetyladriamycin-14-O-hemiadipate (AD 143), a new derivative of adriamycin, on various steps of the enzymic reaction catalyzed by chicken myeloblastosis RNA polymerase II was studied. AD 143 inhibition of RNA synthesis, which was evident at the beginning of the reaction, could not be reversed by increasing the concentrations of any one of the four nucleoside triphosphate substrates of the reaction. Furthermore, the RNA synthesis inhibition was not affected by varying the concentrations of template DNA. The AD 143-induced inhibition caused a reduction of the frequency of RNA chain initiation, whereas the average chain length of RNA synthesized at the end of the reaction remained unaltered. The susceptible step in the initiation process was found to be the formation of stable complexes between RNA polymerase and the DNA template. While AD 143 causes no inhibition of Escherichia coli RNA polymerase activity, it was found not to affect the E. coli RNA polymerase-template DNA complex formation.