Affinity modification of Escherichia coli ribosomes by 4-[(N-2-chloroethyl)N-methylamino]benzyl-5'-phosphamide hexauridylate in a complex stabilized by codon-anticodon interactions on P and A sites

Affinity labeling of E. coli ribosomes with 4-[(N-2-chloroethyl)-N-methylamino] benzyl-5'-phosphamide of hexauridylate was studied within the complex containing tRNAPhe at P site and Phe-tRNAPhe at A site directed by EF-Tu and GTP. Ribosomal proteins as well as rRNA both in 30S and 50S subunits were found to be labelled within the complex. Labeled proteins were identified as S3, S9 and L2. Selectivity of affinity labeling with mRNA analogs was shown to depend on the functional state of the ribosomes. Modification was more selective within the complex stabilized by codon-anticodon interaction both at A and P-sites than within the complex in which this interaction takes place preferentially at P site.     

Selective alkylation of G24 residue in tRNAPhe

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of oligonucleotide d(pAACCA) was studied. G24 residue located near the sequence C17GGDA21 partially complementary to the oligonucleotide moiety of the reagent was shown to be alkylated. Oligonucleotide d(pAACCA) inhibited the alkylation. Association constant of oligonucleotide derivative with tRNAPhe (10(3) M-1) was evaluated from the dependence of the extent of tRNA modification on the concentration of the reagent. The reported method for selective alkylation of tRNA may be used for preparing photoaffinity derivatives of tRNA bearing an arylazidogroups in desired position.     

Structural arrangement of the decoding site of Escherichia coli ribosomes as revealed from the data on affinity labelling of ribosomes by analogs of mRNA--derivatives of oligoribonucleotides

Using derivatives of oligoribonucleotides bearing an active group at the 5'- or 3'-end, the affinity modification of Escherichia coli ribosomes has been investigated in model complexes imitating various steps of initiation and elongation with a different extent of approximation to the real protein-synthesizing system. The protein environment of the ribosome decoding site is determined. The S3, S4, S9, L2, L7/L12 proteins belong to the 5'-region of the decoding site, and the S5, S7, S9, L1, L16 proteins to the 3'-region. In the process of translation the template moves along the external side of the 30 S subunit, from the L1 ridge to the L7/L12 stalk. The structural arrangement of the decoding site or its nearest environment depends on the functional state of ribosomes in the process of translation.     

The proteins of the messenger RNA binding site of Escherichia coli ribosomes.

Oligo(U) derivatives with [14C]-4-(N-2-chloroethyl-N-methylamino)benzaldehyde attached to 3'-end cis-diol group via acetal bond, p(Up)n-1UCHRCl as well as with [14C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to 5'-phosphate via amide bond, ClRCH2NHpU(pU)6 were used to modify 70S E. coli ribosomes near mRNA binding centre. Within ternary complex with ribosome and tRNAPhe all reagents covalently bind to ribosome the extent of modification being 0.1-0.4 mole/mole 70S. p(Up)n-1UCHRCl alkylates either 30S (n=5,7) or both subunits (n=6,8). rRNA is preferentially modified within 30S subunit. ClRCH2NHpU(pU)6 alkylates both subunits the proteins being mainly modified. The distribution of the label among proteins differ for various reagents. S4, S5, S7, S9, S11, S13, S15, S18 and S21 are found to be alkylated within 30S subunit, proteins L1, L2, L6, L7/L12, L19, L31 and L32 are modified in the 50S subunit. Most proteins modified within 30S subunit are located at the "head" of this subunit and proteins modified within 50S subunit are located at the surface of the contact between this subunit and the "head" of 30S subunit at the model of Stoffler.     

Alkylation of tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of d(ATTTTCA)

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of oligonucleotide d(ATTTTCA) complementary to the sequence UGAAms2i6AA psi in the anticodon loop of tRNAPhe was studied. Three guanine residues--G28/29, G24 and G10 were found to be alkylated. Two binding sites for the reagent in the tRNA were assumed to be present. The efficiency of the alkylation of tRNA from these sites as well as an average association constant (Ka 3,8 X 10(3)M-1) for the reagent interaction with tRNA were evaluated.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

Structure of eukaryotic DNA binding sites for steroid hormone-apolipoprotein A-I complexes

High affinity for DNA and synthetic oligonucleotides was detected for apolipoprotein A-I (ApoA-I) by affinity chromatography, affinity modification, and enzymatic analysis. Competitive inhibition and Southern hybridization showed that the tetrahydrocortisol (THC)-ApoA-I complex specifically bound to high-molecular-weight DNA in regions containing GCC/CGG sequences. The CC(GCC)₃ · GG(CGG)₃ duplex was found to be sensitive to nuclease S1 under the action of the THC-ApoA-I complex. The eukaryotic DNA binding sites for steroid (THC, androsterone)-ApoA-I complexes were found to be involved in the initiation of DNA copying in vitro.     

The Effect of Tetrahydrocortisol–Apolipoprotein A-I Complex on the RNA Polymerase Interaction with Eukaryotic DNA and the Rate of Protein Biosynthesis in Hepatocytes

A mechanism of activation of protein biosynthesis in hepatocytes was proposed as effected by the conditioned medium of nonparenchymal liver cells incubated in the presence of high density lipoproteins, cortisol, and lipopolysaccharides. It was found that the increase in the biosynthesis rate was associated with the formation of the tetrahydrocortisol–apolipoprotein A-I (THC–apoA-I) complex in macrophages, which display 5- and 5-reductase activity and are constituents of nonparenchymal liver cell. Using the small-angle X-ray scattering technique, it was shown that the THC–apoA-I–eukaryotic DNA interaction may break hydrogen bonds between pairs of complementary nucleic bases and cause the formation of single-stranded DNA fragments capable of binding to DNA-dependent RNA polymerase. The interaction is highly cooperative and has a saturating mode, up to six enzyme molecules being bound per DNA molecule.     

Interaction of tetrahydrocortisol-apolipoprotein A-I complex with eukaryotic DNA and with single-stranded oligonucleotides

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC-ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (Ka) was 1.66 x 10(6) M-1. Substitution of THC with cortysol considerably decreases the Ka. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.