mRNA-binding site of ribosomes at different stages of translation. II. Affinity modification of Escherichia coli ribosomes bya benzylidene derivative of AUGU6 in pre- and post-translation complexes

Affinity labeling of E. coli ribosomes with the 2',3'-O-[4-(N-2-chloroethyl)-N-methyl-amino]benzylidene derivative of AUGU6 (AUGU6-[14C]CHRCl) was studied within the pretranslocational complex ribosome.AUGU6[14C]CHRCl.tRNA(fMet)(P-site).fMetPhe-tR NA(Phe)(A-site) and posttranslocational complex ribosome.AUGU6[14C]CHRCl.fMetPhe-tRNA(Phe)(P-site). Both 30S and 50S subunits were labeled within these complexes, but the extent of 30S subunit modification was 6-8-fold higher than those for 50S subunit. Ribosomal proteins of both subunits were found to be labeled preferentially. Proteins S1, S5, S11, L1 were identified to be crosslinked with AUGU6[14C]CHRCl within the pretranslocational complex and S7--within the posttranslocational complex from the data of two-dimensional electrophoresis in the polyacrylamide gel.     

Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex

2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.     

Affinity modification of Escherichia coli ribosomes with 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]-benzylidene derivative of AUGU3 in the 70S initiation complexes

Affinity labelling of the Escherichia coli ribosomes with the 2',3'-O-[4-(N-(2-chloroethyl)-N-methylamino]benzylidene derivative of AUGU3(AUGU3[14C]CHRCl) has been studied within 70S initiation complexes ribosome.AUGU3[14C]CHRCl.fMet-tRNA(Metf) and binary complex ribosome.AUGU3[14C]CHRCl. Various ways of the 70S initiation complex formation resulted in differently labelled products. Proteins S5, S7, S9, L1, L16 were thus identified as cross-linked with AUGU3[14C]CHRCl within an initiation complex obtained in the presence of initiation factors IF-1, IF-2, IF-3, whereas only proteins S5 and S7 were cross-linked within the complex obtained with the sole factor IF-2. Proteins S1, S3, L1 and L33 were labelled within the initiation complex obtained nonenzymatically but only protein S1 within the binary complex. In all complexes formed with use of initiation factors labelling of IF-2 factor was invariably observed.     

Interaction of a fluorescent streptomycin derivative with Escherichia coli ribosomes.

The high salt wash of rabbit reticulocyte ribosomes contains two separate factors which can partially reverse the inhibition of polypeptide chain initiation that results when reticulocyte lysate is incubated in the absence of hemin. These two factors, termed initiation factor (IF) 1 and IF-2, have been separated from each other by chromatography on diethylaminoethyl cellulose and then further purified on hydroxyapatite. IF-1 forms a GTP-dependent complex with methionyl-tRNA_f that is retained on Millipore filters. When these factors are added to a system containing reconstituted, salt-extracted ribosomes, IF-1 promotes the binding of methionyl-tRNA_f to the 40 S subunit, whereas IF-2 promotes the formation of 80 S initiation complexes from 40 S complexes. Addition of small amounts of one factor and a saturating level of the other to the unfractionated lysate and incubation in the absence of hemin produce an additive stimulation of protein synthesis. Each factor can also partially reverse the inhibitory effect of the hemin-controlled translational repressor. The implication of these findings for the mechanism of hemin control of protein synthesis in reticulocyte lysates is discussed.     

Affinity modification of Escherichia coli ribosomes with photoactivated analogs of mRNA

Oligoribonucleotide derivatives containing the photoactivated arylazidogroup at 5'-end of the oligonucleotide fragment [2-(N-2,4-dinitro-5-azidophenyl) aminoethyl] phosphamides of the oligoribonucleotides, azido-NH (CH2)2NHpN (pN) n-1, were prepared. It was demonstrated that azido-NH(CH2)2NHpA(pA)4 and azido-NH (CH2)2NHpU (pU)3 stimulate the binding of the codonspecific aminoacyl-tRNA with ribosome. After irradiation of the ternary complex ribosome-azido-NH (CH2)2NHpU (pU) n-1 X tRNA with UV-light (lambda greater than 350 nm) covalent binding of the reagent to ribosome occurs. Up to 10% of the reagent, bound in the ternary complex with ribosome, is cross-linked with the ribosomal proteins of 30S and 50S subunits. The ribosomal RNA are not modified by azido-NH (CH2)2NHpU (pU) n-1. The proteins of 30S and 50S subunits, modified with azido-NH (CH2)2NHpU (pU) n-1 with n = 4,7 and 8, were identified. It is shown that proteins of 30S subunits S3, S4, S9, S11, S12, S14, S17, S19, S20 undergo modification. The proteins of 50S subunits L2, L13, L16, L27, L32, L33 are modified. The set of the modified proteins essentially depends on the length of the oligonucleotide part of the reagent and on occupancy of ribosome A-site by a molecule of tRNA.     

Affinity modification of Escherichia coli ribosomes near the acceptor tRNA-binding site

It was shown that Phe-tRNA Phe derivatives bearing arylazidogroups scattered statistically on N7 guanosine residues retain the ability to EF-Tu-dependent binding to E. coli ribosomes. UV-irradiation of the corresponding complex with the derivative of Phe-tRNA Phe located at A-site results in a specific modification of both ribosomal subunits to an approximately equal extent. It was found that proteins S9, S15, S16, S17, S18, S19 and L8/L9, L13, L15, L27 are labelled at A-site.     

Copies of protein S6 in Escherichia coli ribosomes

A reinvestigation of the copy number of protein S6 in Escherichia coli ribosomes shows that there is only one copy of S6 in 30 S subunits and in 70 S ribosomes. The previously reported higher value (Subramanian, A. R. (1980) J. Biol. Chem. 255, 6941-6946) is shown to arise from the presence in the usual ribosome preparations of a protein which co-electrophoreses with S6 but does not react with S6 antiserum. This protein is removed when ribosomes are purified by passage through a sucrose gradient.     

Affinity labelling of a partially purified ecdysteroid receptor with a bromoacetylated 20-OH-ecdysone derivative

The novel bromoacetyl ecdysteroid IV, (20R,22R)-2 beta,3 beta,14 alpha,20,22,25 xi-hexahydroxy-26-(3- bromoacetoxypropyl)-5 beta-cholest-7-en-6-one, BAEIV, has been synthesized by extending the side chain on C26 of 20-OH-ecdysone. BAEIV meets all the requirements for an affinity-labelling reagent. It reacts with the partially purified ecdysteroid receptors of Drosophila melanogaster rapidly and almost quantitatively. Reactions require only micromolar concentrations of BAEIV. The rate of the affinity-labelling reaction is determined by the association of BAEIV with the ecdysteroid receptor. The value of the apparent reaction rate constant is very similar to that of the association rate constant for the binding of 20-OH-ecdysone to the ecdysteroid receptor. Product analysis of the reaction of [14C]BAEIV with the ecdysteroid receptor revealed two labelled peptides having molecular masses 150 kDa and 90 kDa. The smaller peptide is possibly a proteolytic fragment of the larger peptide. The identification of a 150-kDa peptide by chemical affinity labelling of the ecdysteroid receptor agrees with previously reported photoaffinity-labelling results from our laboratory. The results also demonstrate that the ecdysteroid receptor of D. melanogaster has a molecular mass higher than all other vertebrate steroid hormone receptors studied so far.     

A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes.

A photoaffinity labelling study of the messenger RNA-binding region of E. coli ribosomes has been made, using oligoadenylic acids as mRNA analogs. The oligonucleotides, of chain length 6 to 8 and thus several nucleotides longer than oligonucleotides previously employed for this purpose, carried a radioactive photolabile aromatic azide reagent bound covalently to the 3'-terminal ribose moiety. The synthesis of the reagent, p-azidobenzoyl-(3H)-glycylhydrazide, is described. The derivatized oligonucleotides were shown to be functional messengers. They stimulated the binding of the cognate aminoacyl-tRNA, lysyl-tRNA: their binding was reciprocally stimulated by lysyl-tRNA; and they competed with underivatized oligoadenylates for ribosomal binding sites. When the 70 S ribosomal binding complex was irradiated, the photolabile reagent reacted covalently with both RNA and proteins of the 30 S subunit and with tRNA, but not with the 50 S subunit. The 16 S RNA appeared to be labelled at more than one site. Of the proteins, S3 and S5 reacted with the reagent with high specificity; and the possibility was not eliminated that S4 may have been labelled to a minor degree. Functional studies in other laboratories have implicated S3 and S5 in the decoding process, but these proteins were not labelled by any of the previously reported mRNA affinity labelling analogs. The results reported here therefore indicate that S3 and S5 not only affect the decoding process, but are located in the mRNA-binding region of the ribosome, presumably to the 3' side of the decoding site.     

Affinity labeling at the A-site of Escherichia coli ribosomes by a non-hydrolyzable gamma-amide analog of GTP

gamma-Amides of GTP and affinity and photoaffinity derivatives of gamma-amides of GTP: gamma-anilide of GTP, gamma-(4-azido)anilide of GTP, gamma-[N-(4-azidobenzyl)-N-methyl]amide of GTP, gamma[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP and gamma-[4-N-(2-oxoethyl)-N-methylaminobenzyl]amide of GTP substituted efficiently for GTP in the EF-Tu-dependent transfer of aminoacyl-tRNA to the ribosome but, in contrast to GTP, they were not hydrolyzed in this process. They represent a new class of non-hydrolyzable GTP analogs with preserved gamma-phosphodiester bond. The radioactive analog of GTP: gamma-[4-N-(2-chloroethyl)-N-methylamino[14C]benzyl]amide of GTP was used as an affinity labeling probe for the identification of components of the GTPase center formed in the EF-Tu-dependent transfer reaction of aminoacyl-tRNA to the ribosomal A-site. Within a six-component complex of poly(U)-programmed E. coli ribosomes with elongation factor Tu, Phe-tRNA(Phe) (at the A-site), tRNA(Phe) (at the P-site) and the [14C]GTP analog, mainly the ribosomal 23S RNA and to a lesser extent the ribosomal proteins L17, L21, S16, S21 and the ribosomal 16S RNA were labeled by the reagent. No significant modification of EF-Tu was detected.     

Fluorescence studies of the binding of a yeast tRNAPhe derivative to Escherichia coli ribosomes.

The equilibrium binding of a highly fluorescent derivative of yeast tRNA^Phe to Escherichia coli 70 S ribosomes was studied fluorimetrically at 7 °C in 25 mm-magnesium. Under these conditions 70 S ribosomes bind two deacylated tRNAs stoichiometrically. An analysis of the binding data using a model in which occupancy of the weaker site requires prior occupancy of the stronger site leads to apparent association constants of (1.00 ± 0.05) × 10⁹m^?1 and (3.4 ± 0.2) × 10⁷m^?1. The use of an independent site model does not change these values appreciably. The observed binding constants do not depend upon the presence or absence of the messenger RNA, poly(U). However, spectroscopic evidence strongly suggests that the anticodons of both bound tRNAs are in contact with the message. This evidence further suggests that in the presence of poly(U) the environment of the hypermodified base adjacent to the anticodon is substantially different in the two sites. This may reflect a difference in the conformation of the anticodon loops or an interaction between the hypermodified base of the weak site tRNA and the anticodon loop of the strong site tRNA.     

Interaction of bovine mitochondrial ribosomes with Escherichia coli initiation factor 3 (IF3)

Mammalian mitochondrial ribosomes are distinguished from their bacterial and eukaryotic-cytoplasmic counterparts, as well as from mitochondrial ribosomes of lower eukaryotes, by their physical and chemical properties and their high protein content. However, they do share more functional homologies with bacterial ribosomes than with cytoplasmic ribosomes. To search for possible homologies between mammalian mitochondrial ribosomes and bacterial ribosomes at the level of initiation factor binding sites, we studied the interaction of Escherichia coli initiation factor 3 (IF3) with bovine mitochondrial ribosomes. Bacterial IF3 was found to bind to the small subunit of bovine mitochondrial ribosomes with an affinity of the same order of magnitude as that for bacterial ribosomes, suggesting that most of the functional groups contributing to the IF3 binding site in bacterial ribosomes are conserved in mitochondrial ribosomes. Increasing ionic strength affects binding to both ribosomes similarly and suggests a large electrostatic contribution to the reaction. Furthermore, bacterial IF3 inhibits the Mg2+-dependent association of mitochondrial ribosomal subunits, suggesting that the bacterial IF3 binds to mitochondrial small subunits in a functional way.