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.     

Neighbourhood of the central fold of the tRNA molecule bound to the E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop.

The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.     

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.     

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.     

Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives

A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).     

Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome

The 3'-terminal -A-C-C-A sequence of yeast tRNA(Phe) has been modified by replacing either adenosine-73 or adenosine-76 with the photoreactive analogue 8-azidoadenosine (8N3A). The incorporation of 8N3A into tRNA(Phe) was accomplished by ligation of 8-azidoadenosine 3',5'-bisphosphate to the 3' end of tRNA molecules which were shortened by either one or four nucleotides. Replacement of the 3'-terminal A76 with 8N3A completely blocked aminoacylation of the tRNA. In contrast, the replacement of A73 with 8N3A has virtually no effect on the aminoacylation of tRNA(Phe). Neither substitution hindered binding of the modified tRNAs to Escherichia coli ribosomes in the presence of poly(U). Photoreactive tRNA derivatives bound noncovalently to the ribosomal P site were cross-linked to the 50S subunit upon irradiation at 300 nm. Nonaminoacylated tRNA(Phe) containing 8N3A at either position 73 or position 76 cross-linked exclusively to protein L27. When N-acetylphenylalanyl-tRNA(Phe) containing 8N3A at position 73 was bound to the P site and irradiated, 23S rRNA was the main ribosomal component labeled, while smaller amounts of the tRNA were cross-linked to proteins L27 and L2. Differences in the labeling pattern of nonaminoacylated and aminoacylated tRNA(Phe) containing 8N3A in position 73 suggest that the aminoacyl moiety may play an important role in the proper positioning of the 3' end of tRNA in the ribosomal P site. More generally, the results demonstrate the utility of 8N3A-substituted tRNA probes for the specific labeling of ribosomal components at the peptidyltransferase center.     

Identification of nucleotide residues essential for RNase P activity from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg(2+) ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.     

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.     

Transit of tRNA through the Escherichia coli ribosome. Cross-linking of the 3' end of tRNA to specific nucleotides of the 23 S ribosomal RNA at the A, P, and E sites

When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.     

Photoaffinity modification of Escherichia coli ribosomes by fMet-tRNAf Met derivatives in the 70S initiation complex

Photoaffinity labeling of E. coli ribosomes within the 70S initiation complex was studied by using photoreactive derivatives of fMet-tRNAfMet bearing arylazidogroups scattered statistically over guanosine residues. It is shown that fMet-azido-tRNAfMet-II bearing 2 moles of the reagent residues per mole of tRNA (modified in the conditions of stability of tRNA tertiary structure) is fully active in aminoacylation and in the factor-dependent binding with ribosomes to form the 70S initiation complex. Functional activity of fMet-azido-tRNAfMet-I bearing also 2 moles of the reagent residues per mole of tRNA (but modified in conditions of lability of tRNA tertiary structure) decreases up to approximately 45% in aminoacylation and up to 70% in IF-2 X GTP-dependent binding to the ribosomes. Irradiation of complexes 70S ribosome-MS2-RNA-fMet-azido-tRNAfMet results in covalent linking of the tRNA derivative to the ribosomes. Both subunits are labeled, the 30S to a larger extent than 50S. It is shown that fMet-azido-tRNAfMet-II labels proteins S1, S7, S9, L27 whereas fMet-azido-tRNAfMet-1--proteins S1, S3, S5, S9, S14, L1, L2, L7/L12.     

The 5S rRNA-protein complex of Escherichia coli studied by carbodiimide modification]

5S rRNA-protein complex has been reconstituted from 5S rRNA and total protein of large (L) ribosomal subunit of Escherichia coli. The complex consists of 5S rRNA and 3 proteins only: L5, L18, L25. A water-soluble carbodiimide [N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-methyl-p-toluolsulp honate] cross-links L18 to 5S rRNA at pH 7.2 and L25 to 5S rRNA at pH 7.7. This pH-dependence of cross-linked proteins is a consequence of the difference in stability of the initial complex: the complex has all three proteins at pH 7.7 but L18 mainly at pH 7.2. It has been shown that L18 stimulates the chemical modification of U87 and U89 residues of 5S rRNA by carbodiimide. A model of L18-5S rRNA complex has been proposed.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift

Frameshift mutations can be suppressed by a variety of differently acting external suppressors. The +1 frameshift mutation hisC3072, which has an extra G in a run of Gs, is corrected by the external suppressor mutation sufF44. We have shown that sufF44 and five additional allelic suppressor mutations are located in the gene argU coding for the minor tRNAArgmnm5UCU and alter the secondary and/or tertiary structure of this tRNA. The C61U, G53A, and C32U mutations influence the stability, whereas the C56U, C61U, G53A, and G39A mutations decrease the arginylation of tRNAArgmnm5UCU. The T-10C mutant has a base substitution in the -10 consensus sequence of the argU promoter that reduces threefold the synthesis of tRNAArgmnm5UCU . The lower amount of tRNAArgmnm5UCU or impaired arginylation, either independently or in conjunction, results in inefficient reading of the cognate AGA codon that, in turn, induces frameshifts. According to the sequence of the peptide produced from the suppressed -GGG-GAA-AGA- frameshift site, the frameshifting tRNA in the argU mutants is tRNAGlumnm5s2UUC, which decodes the GAA codon located upstream of the AGA arginine codon, and not the mutated tRNAArgmnm5UCU. We propose that an inefficient decoding of the AGA codon by a defective tRNAArgmnm5UCU stalls the ribosome at the A-site codon allowing the wild-type form of peptidyl-tRNAGlumnm5s2UUC to slip forward 1 nucleotide and thereby re-establish the ribosome in the 0-frame. Similar frame-shifting events could be the main cause of various phenotypes associated with environmental or genetically induced changes in the levels of aminoacylated tRNA.     

Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome.

A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.     

The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.     

Contacts of ribosomal proteins with tRNAPhe and 16S RNA in analogs of the 30S initiation complex

Direct RNA-protein contacts have been studied by means of ultraviolet-induced (254 nm) cross-links inside complexes of NAcPhe-tRNAPhe, Phe-tRNAPhe and deacylated tRNAPhe with poly(U)-charged 30S subunit of Escherichia coli ribosome. In the first two complexes tRNA directly contacts with the similar sets of proteins (S4, S5, S7, S9/S11; S6 and S8 are found only in the second complex). These sets are similar to that in the fMet-tRNAfMet X 30S X mRNA complex, evidencing similar disposition of tRNAs in these three complexes. 16S RNA contacts in free 30S subunit mainly with proteins S4, S7 and S9/S11. In both complexes, containing NAcPhe-tRNAPhe and Phe-tRNAPhe, 16S RNA contacts with essentially the same proteins (S4, S5, S7, S8, S9/S11, S10, S15, S16 and S17) and in the same ratio, evidencing similar conformation of 30S subunit in these two complexes. In the third complex deacylated tRNAPhe contacts with proteins S4, S5, S6, S8, S9/S11 and S15, 16S RNA-protein interaction differs from those in the first two complexes by a remarkable decrease of cross-linked proteins S8, and S9/S11 and by the appearance of a large amount of cross-linked proteins(s) S13/S14. Hence, this complex differs from the first two by conformation of 30S subunit and, probably, by disposition and/or conformation of tRNA.     

Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm

Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.     

Effects of modification of 4-thiouridine in E. coli tRNA(fMet) on its methyl acceptor activity by thermostable Gm-methylases

tRNA(guanosine-2'-)-methyltransferases (Gm-methylases) isolated from extreme thermophiles, Thermus thermophilus strains HB 27 and HB 8, methylate the 2'-OH of the G18 ribose of the GG sequence in the D loop of tRNA, by recognizing the D "loop-stem" structure as a minimal requirement. To examine the role of the consensus uridine residue at position 8 (U8) adjacent to the D "loop-stem" region in the recognition of Gm-methylase, 4-thiouridine at this position (s4U8) in Escherichia coli tRNAfMet was modified reversibly with S-benzylthioisothiourea (sBTIU) or irreversibly by UV light. The initial velocities of the methylation reaction for the sBTIU-modified and the UV-induced cross-linked tRNAs were decreased to 40 and 30%, respectively, of that of the intact tRNA, but the sBTIU-modified tRNA regained almost full activity on reduction with beta-mercaptoethanol. Although both of the modified tRNAfMetS showed larger Km (although to different extents) and slightly smaller Vmax than the intact tRNAfMet, they retained full activities of methylation with tRNA(adenine-1-)-methyltransferase (m1A-methylase) and of aminoacylation with aminoacyl-tRNA synthetase (ARS) fraction as well, both of which were prepared from T. thermophilus strain HB 27. The 5'-half fragments derived from the sBTIU-modified and cross-linked tRNAfMetS showed methylation efficiency (Vmax/Km) not appreciably different from that of the unmodified 5'-half fragment. These results suggest that the conformation of S4U8 residue of tRNA is deeply involved in the recognition of tRNA by Gm-methylase.     

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.     

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).