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.     

Polynucleotide-protein interactions in the translation system. Identification of proteins interacting with tRNA in the A- and P-sites of E. coli ribosomes.

Ultraviolet irradiation (lambda = 254 nm) of ternary complexes of E. coli 70 S ribosomes with poly(U) and either Phe-tRNAPhe (in the A-site) or NAcPhe-tRNAPhe (in the P-site) effectively induces covalent linking of tRNA with a limited number of ribosomal proteins. The data obtained indicate that in both sites tRNA is in contact with proteins of both 30 S and 50 S subunits (S5, S7, S9, S10, L2, L6 and L16 proteins in the A-site and S7, S9, S11, L2, L4, L7/L12 and L27 proteins in the P-site). Similar sets of proteins are in contact with total aminoacyl-tRNA and N-acetylaminoacyl-tRNA. However, here no contacts of tRNA in the P-site with the S7 and L25/S17 proteins were revealed, whereas in the A-site total aminoacyl-tRNA contacts L7/L12. Proteins S9, L2 and, probably, S7 and L7/L12 are common to both sites.     

Affinity modification of Escherichia coli ribosomes with the non-hydrolyzed GTP analog, gamma-[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP

Substituted gamma-amides of GTP viz. GTP gamma-[4-N-(2-chloro- and gamma-[4-N-(2-hydroxyethyl)-N-methylaminobenzyl]amide (CIRCH2NHpppG and OHRCH2NHpppG, resp.) were shown to be unhydrolisable GTP analogues in the EF-Tu-dependent GTP-ase reaction of ribosomes. The reactive analogue, CIRCH2NHpppG, was used for affinity labelling within the 70S ribosome.poly(U).tRNAPhe(P-site).Phe-tRNAPhe.EF-Tu.CIR[14C]CH2.NHpppG complex. Both 50S and 30S subunits were thus labelled but 50S subunit was modified considerably more than 30C subunit. Labelled were proteins L17, L21, S16, S21, and rRNA of both subunits, 23C rRNA within 50C subunit being labelled preferentially as compared with 50C proteins. No labelling of EF-Tu within the complex was detected.     

The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.     

Properties of elongation factor G: its interaction with the ribosomal peptidyl-site

EF-G bound to poly(U)·ribosomes prevents enzymatic or nonenzymatic binding of charged tRNA not only to the A-site but also to the P-site. In turn, charged tRNA bound either to the P- or A-site prevents formation of EF-G·GMPPCP·ribosome complex. Ribosomes carrying newly synthetized peptidyl-tRNA in pretranslocative state are also unable to form stable complexes with EF-G. The functional implications of these observations are discussed and it is suggested that tRNA plays a regulatory role in the interaction of EF-G with ribosomes during the cyclic process of elongation.     

Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.     

The effect of tRNA binding on the structure of 5 S RNA in Escherichia coli. A chemical modification study

The structure of 5 S RNA within the 70 S ribosome from Escherichia coli was studied using the chemical reagent kethoxal (alpha-keto-beta-ethoxybutyraldehyde) to modify accessible guanosines. The modification pattern of 5 S RNA from free 70 S ribosomes was compared with that of poly(U) programmed ribosomes where tRNA had been bound to both the A- and P-sites. Binding to the ribosomal A-site was achieved enzymatically using the elongation factor Tu and GTP in the presence of deacylated tRNA which blocks the ribosomal P-site. Modified guanosines were identified after partial RNase T1 hydrolysis and separation of the hydrolysis products on sequencing gels. Binding of tRNA to the ribosome leads to a strong protection of 5 S RNA guanosine G-41 and to some degree G-44 from kethoxal modification. The limited RNase T1 hydrolysis pattern provides evidence for the existence of a 5 S RNA conformation different from the known 5 S RNA A- and B-forms which are characterized by their gel electrophoretic mobility. The importance of 5 S RNA for the binding of tRNA to the ribosome is discussed.     

Protein environment of the sense codon of the template in the A site of the human ribosome as inferred from crosslinking to oligoribonucleotide derivatives

The protein environment of each nucleotide of the template codon located in the A site of the human ribosome was studied with UUCUCAA and UUUGUU derivatives containing a Phe codon (UUC and UUU, respectively) and a perfluoroarylazido group at U4, U5, or U6. The analogs were positioned in the ribosome with the use of tRNA(Phe), which is cognate to the UUC or UUU codon and directs it to the P site, bringing a modified codon in the A site with a modified nucleotide occupying position +4, +5, or +6 relative to the first nucleotide of the P-site codon. On irradiation of ribosome complexes with tRNA(Phe) and mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins to a greater extent than the rRNA. The 18S rRNA nucleotides crosslinking to the analogs were identified previously. Of the small-subunit proteins, S3 and S15 were the major targets of modification in all cases. The former was modified both in ternary complexes and in the absence of tRNA, and the latter, only in ternary complexes. The extent of crosslinking of mRNA analogs to S15 decreased when the modified nucleotide was shifted from position +4 to position +6. The results were collated with the data on ribosomal proteins located at the decoding site of the 70S ribosome, and conclusion was made that the protein environment of the A-site codon strikingly differs between bacterial and eukaryotic ribosomes.     

Ribosomal proteins S7 and L1 are located close to the decoding site of E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3''-end of the anticodon.

Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.     

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.     

Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyl-tRNA molecular rulers.

A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe. Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.     

Codon-specific interaction of uncharged transfer-RNA with eukaryotic ribosomes.

Rat liver ribosomes bound [32P]tRNAPhe in both a codon-dependent and codon-independent manner. The codon-dependent binding was studied further by utilising the ability of the unchanged tRNAPhe to inhibit the poly(U)-directed binding of [3H]Phe-tRNA to ribosomes. At least part of the codon-dependent binding of uncharged tRNA appears to be to the ribosomal A-site.     

Template location on the human ribosome: environment of the mRNA nucleotide adjacent to the A-site codon on the 3'-side

The 18S rRNA nucleotides close to the 80S ribosome template nucleotide adjacent to the A-site codon on the 3-end (i.e., the nucleotide in position +7 relative to the first nucleotide of the P-site codon) were identified using template-controlled chemical affinity ligation. For this purpose, used the photoreactive mRNA analogues with a perfluorophenylazido group attached through various linkers to the uridine C5,3'-terminal phosphate, or guanosine N7 were used. The position of the mRNA analogues on the ribosome was preset using tRNAPhe, which recognized the phenylalanine codon directed to the P-site. An analysis of the rRNAs isolated from the irradiated complexes of 80S ribosomes showed that all the analogues are almost equally ligated to the 18S rRNA nucleotides we attributed to the A-site codon environment: namely, to nucleotides A1823, A1824, and A1825 of the 3'-minidomain and to the 620-630 fragment of the 18S rRNA 5'-domain. In addition, we identified a new component of the mRNA binding site of human ribosomes, nucleotide C1698 belonging to the 18S rRNA 3-minidomain, using analogues bearing a perfluorophenylazido group on uridine and guanine residues. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains.

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA-ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNA(Phe) anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNA(Phe), ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared. Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNA(Phe) enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with KpS approximately 50-60 nM, were afforded significantly greater protection by both yeast tRNA(Phe) and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (KpS approximately 100-200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33. ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33. However, protection of G926 and G1338 (KpS between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (KpS = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other.     

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

Direct cross-linking of heptauridilate to E. coli ribosomes by water-soluble carbodiimide in the complex stabilized by codon-anticodon interaction at both A- and P-sites

Affinity labelling of E. coli ribosomes is performed by treatment with water-soluble carbodiimide of the complex of ribosomes with (pU)7, tRNAPhe at the P-site and with Phe-tRNAPhe (complex I) and without Phe-tRNAPhe (complex II) at the A-site. The extent of modification is, respectively, 0.06 and 0.026 mol (pU)7 per mol ribosomes. Protein S3 is found as a single labelled protein in complex I, whereas S7, S8, L25 are modified in complex II. Thus, in the absence of a large spacer group within the complex stabilized by codon-anticodon interactions at both A- and P-sites, a highly selective modification occurs.     

Protein Environment of the Sense Codon of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking to Oligoribonucleotide Derivatives

The protein environment of each nucleotide of the template codon located in the A site of the human ribosome was studied with UUCUCAA and UUUGUU derivatives containing a Phe codon (UUC and UUU, respectively) and a perfluoroarylazido group at U4, U5, or U6. The analogs were positioned in the ribosome with the use of tRNA^Phe, which is cognate to the UUC or UUU codon and directs it to the P site, bringing a modified codon in the A site with a modified nucleotide occupying position +4, +5, or +6 relative to the first nucleotide of the P-site codon. On irradiation of ribosome complexes with tRNA^Phe and mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins to a greater extent than the rRNA. The 18S rRNA nucleotides crosslinking to the analogs were identified previously. Of the small-subunit proteins, S3 and S15 were the major targets of modification in all cases. The former was modified both in ternary complexes and in the absence of tRNA, and the latter, only in ternary complexes. The extent of crosslinking of mRNA analogs to S15 decreased when the modified nucleotide was shifted from position +4 to position +6. The results were collated with the data on ribosomal proteins located at the decoding site of the 70S ribosome, and conclusion was made that the protein environment of the A-site codon strikingly differs between bacterial and eukaryotic ribosomes.     

Interactions of yeast tRNAPhe with ribosomes from yeast and Escherichia coli. A fluorescence spectroscopic study.

The interaction of ethidium-labeled tRNAPhe from yeast with ribosomes from yeast and Escherichia coli was studied by stead-state measurements of fluorescence intensity and polarization. The ethidium label was covalently inserted into either the anticodon or the dihydrouridine loop of the tRNA. The codon-independent formation of a tRNA-ribosome complex led to only a moderate increase of the observed fluorescence polarization indicating a considerable internal mobility of the labeled parts of the tRNA molecule in the ribosome complex. When the ribosome complex was formed in the presence of poly(U), the probes both in the dihydrouridine loop and in the anticodon loop were strongly immobilized, the latter exhibiting a substantial increase in fluorescence intensity. A smaller intensity change was observed when E. coli ribosomes were used, although the extent of immobilization was found to be similar in this case. Competition experiments with non-labeled tRNAPhe showed that the labeled tRNAPheEtd was readily released from the complex with yeast ribosomes when poly(U) was absent, whereas in the presence of poly(U) it was bound practically irreversibly. The finding that the mobility of a probe in the dihydrouridine loop is affected by the codon-anticodon interaction on the ribosome suggests a conformational change of the ribosome-bound tRNA which may involve opening of the tertiary structure interactions between the dihydrouridine and the TpsiC loop.     

Direct tRNA-protein interactions in ribosomal complexes.

Nucleotide residues in E. coli tRNA(Phe) interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by UV-induced cross-linking. In the tRNA(Phe) molecule located in the Ab-site (pretranslocated complex) residues A9, G18, A26 and U59 are cross-linked with proteins S10, L27, S7 and L2, respectively. In tRNA(Phe) located in the Pt-site (posttranslocated complex) residues C17, G44, C56 and U60 are cross-linked with proteins L2, L5, L27 and S9, respectively. The same cross-links (except for G44-L5) have been found for tRNA in the Pb-site of the pretranslocated ribosomal complex. None of the tRNA(Phe) residues cross-linked with proteins in the complexes examined by us are involved in the stabilization of the secondary structure, but residues A9, G18, A26, G44 and C56 participate in stabilization of tRNA tertiary structure. Since translocation of tRNA(Phe) from Ab- to P-site is accompanied by changes of tRNA contacts with proteins L2 and L27, we postulate that this translocation is coupled with tRNA turn around the axis joining the anticodon loop with the CCA-end of the molecule. This is in agreement with the idea about the presence of a kink in mRNA between codons located in the ribosomal A- and P-sites. In all E. coli tRNAs with known primary structure positions 18 and 56, interacting with L27 protein, when tRNA is located either in A- or P-site, are invariant, whereas positions 17 and 60, interacting with proteins only when tRNA is in the P-site, are strongly conserved. In positions 9, 26 and 59 purines are the preferred residues. In most E. coli tRNAs deviations from the consensus in these three positions is strongly correlated.     

Codon-anticodon interaction at the ribosomal E site

The question of whether or not the tRNA at the third ribosomal binding site specific for deacylated tRNA (E site) undergoes codon-anticodon interaction was analyzed as follows. Poly(U)-programmed ribosomes each carrying two [14C]tRNAPhe molecules were subjected to a chasing experiment using various tRNA species. At 0 degree C Ac[3H]Phe-tRNAPhe did not trigger any chasing whereas deacylated cognate tRNAPhe provoked a strong effect; non-cognate tRNALys was totally ineffective. This indicates that the second [14C]tRNAPhe cannot be present at the A site but rather at the E site (confirming previous observations). In the presence of poly(U) or poly(A) ribosomes bound the cognate tRNA practically exclusively as second deacylated tRNA, i.e. [14C]tRNAPhe and [14C]tRNALys, respectively. Thus, the second deacylated tRNA binds in a codon-dependent manner. [14C]tRNALys at the P site and Ac[3H]Lys-tRNALys at the A site of poly(A)-primed ribosomes were translocated to the E and P sites, respectively, by means of elongation factor G. The E site-bound [14C]tRNALys could be significantly chased by cognate tRNALys but not by non-cognate tRNAPhe, indicating the coded nature of the E site binding. Additional evidence is presented that the ribosome accommodates two adjacent codon-anticodon interactions at either A and P or P and E sites.