Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center

Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.     

Photoaffinity modification of Escherichia coli ribosomes near the tRNA-binding centers by tRNAPhe derivatives carrying arylazido groups on guanosine residues

Photoreactive derivatives of tRNAPhe (E. coli) bearing arylazido groups scattered statistically over guanosine residues (azido-tRNA) were applied for affinity labelling of E. coli ribosomes in elongation factor-dependent or factor-free model systems mimicking different steps of elongation. It is shown that UV-irradiation of the corresponding complexes of ribosomes with tRNA derivatives results in labelling of both subunits, the 30S one is labelled preferentially. In all experiments only ribosomal proteins were labelled. Comparison of the sets of proteins labelled by tRNA derivatives in different states at P-site allowed us to draw important conclusions concerning the influence of peptidyl moiety and of the occupancy of the A-site with aminoacyl- or peptidyl-tRNA on the arrangement of tRNA located at the P-site. Three of the 30S proteins--S11, S13 S19--are labelled with tRNA derivatives located at P-site in all states.     

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.     

tRNA-binding centers of Escherichia coli ribosomes and their structural organization

Problems concerning the interaction of tRNA with Escherichia coli ribosomes in different functional states were studied. These problems deal first of all with the number of tRNA-binding sites on ribosome, the conservation of the codon-anticodon interaction at the P-site and with regions of tRNA interacting with ribosome. The problems concerning structural organization of tRNA-binding centers are discussed in more detail.     

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.     

Localization of the elongation factor Tu binding site on Escherichia coli ribosomes

Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.     

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.     

Puromycin interacts with the donor (P) site of Escherichia coli ribosomes

Puromycin inhibits the interaction of peptidyl-tRNA analogs AcPhe-tRNA Phe ox-red, AcPhe-tRNA Phe and FMet-tRNA f Met with the donor (P) site of Escherichia coli ribosomes. It affects both template-free and poly(U)-dependent systems. The inhibition is apparently due to direct competition for the P-site. On isolated 30S ribosomal subunits it was shown that the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements of the inhibition revealed that the affinity constant of puromycin for the P-site is not less than its affinity for the A-moiety of the peptidyl transferase center [1.1 divided by 3.8) X 10(3) M-1).     

Puromycin binding to the donor (P-) site of Escherichia coli ribosomes

Puromycin inhibits the interaction of peptidyl-tRNA analogues AcPhe-tRNA_ox-red^Phe, AcPhe-tRNA^Phe and fMet-tRNA_f^Met with the donor (P-) site of Escherichia coli ribosomes. affects almost equally both the rate of the binding and the equilibrium of the system. This means that the effect is due to direct competition for the P-site, but not due to the indirect influence via the acceptor (A-) site. The inhibition was observed also in 30 S ribosomal subunits, therefore the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements show that the affinity of puromycin for its new ribosomal binding site is similar to its affinity for the acceptor site of the peptidyl transferase center.     

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.     

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe

Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].     

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.     

Ribonuclease sensitivity of Escherichia coli ribosomes

Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099-1110. 1966.-The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10(-4)m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities.