Jean Pierre Ebel

Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase: A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3 or 5-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5 side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.

Use of Lead(II) to Probe the Structure of Large RNA's. Conformation of the 3′ Terminal Domain of E. coli 16S rRNA and its Involvement in Building the tRNA Binding Sites

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNAs and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3 domain of the E. coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3 end of the RNA. The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections. A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.

Comparison of the tertiary structure of yeast tRNAAsp and tRNAPhe in solution: Chemical modification study of the bases☆

A comparative study of the solution structures of yeast tRNA(Asp) and tRNA(Phe) was undertaken with chemical reagents as structural probes. The reactivity of N-7 positions in guanine and adenine residues was assayed with dimethylsulphate and diethyl-pyrocarbonate, respectively, and that of the N-3 position in cytosine residues with dimethylsulphate. Experiments involved statistical modifications of end-labelled tRNAs, followed by splitting at modified positions. The resulting end-labelled oligonucleotides were resolved on polyacrylamide sequencing gels and analysed by autoradiography. Three different experimental conditions were used to follow the progressive denaturation of the two tRNAs. Experiments were done in parallel on tRNA(Asp) and tRNA(Phe) to enable comparison between the two solution structures and to correlate the results with the crystalline conformations of both molecules. Structural differences were detected for G4, G45, G71 and A21: G4 and A21 are reactive in tRNA(Asp) and protected in tRNA(Phe), while G45 and G71 are protected in tRNA(Asp) and reactive in tRNA(Phe). For the N-7 atom of A21, the different reactivity is correlated with the variable variable loop structures in the two tRNAs; in the case of G45 the results are explained by a different stacking of A9 between G45 and residue 46. For G4 and G71, the differential reactivities are linked to a different stacking in both tRNAs. This observation is of general significance for helical stems. If the previous results could be fully explained by the crystal structures, unexpected similarities in solution were found for N-3 alkylation of C56 in the T-loop, which according to crystallography should be reactive in tRNA(Asp). The apparent discrepancy is due to conformational differences between crystalline and solution tRNA(Asp) at the level of the D and T-loop contacts, linked to long-distance effects induced by the quasi-self-complementary anticodon GUC, which favour duplex formation within the crystal, contrarily to solution conditions where the tRNA is essentially in its free state.

Target Site of Escherichia coli Ribosomal Protein S15 on its Messenger RNA Conformation and Interaction with the Protein

The regulatory site of ribosomal protein S15 has been located in the 5 non-coding region of the messenger, overlapping with the ribosome loading site. The conformation of an in vitro synthesized mRNA fragment, covering the 105 nucleotides upstream from the initiation codon and the four first codons of protein S15, has been monitored using chemical probes and RNase V1. Our results show that the RNA is organized into three domains. Domains I and II, located in the 5 part of the mRNA transcript, are folded into stable stem-loop structures. The 3-terminal domain (III), which contains the Shine-Dalgarno sequence and the AUG initiation codon, appears to adopt alternative conformations. One of them corresponds to a rather unstable stem-loop structure in which the Shine-Dalgarno sequence is paired. An alternative potential structure involves a pseudo-knot interaction between bases of this domain and bases in the loop of domain II. The conformation of several RNA variants has also been investigated. The deletion of the 5-proximal stem-loop structure (domain I), which has no effect on the regulation, does not perturb the conformation of the two other domains. The deletion of domain II, leading to a loss of regulatory control, prevents the formation of the potential helix involved in the pseudo-knot structure and results in a stabilization of the alternative stem-loop structure in domain III. The replacement of another base in domain III involved in pairing in the two alternative structures mentioned above should induce a destabilization of both structures and results in a loss of the translational control. However, the replacement of another base in domain III, which does not abolish the control, results in the loss of the conformational heterogeneity in this domain and yields a stable conformation corresponding to the pseudo-knot structure. Thus, it appears that any mutation that disrupts or alters the formation of the pseudo-knot impairs the regulatory mechanism. Footprinting experiments show that protein S15 is able to bind to the synthesized fragment and provide evidence that the protein triggers the formation of the pseudo-knot conformation. A mechanism can be postulated in which the regulatory protein stabilizes this particular structure, thus impeding ribosome initiation.

A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

Abstract The hydrolysis of several aminoacylated transfer RNAs, by double-strand-specific ribonuclease from Naja oxiana was studied. The sensitivity to this enzyme of Phe-tRNAPhe, Glu-tRNAGlu and Met-tRNAmMet from Escherichia coli and Phe-tRNAPhe from yeast was examined, both in the free state and complexed to E. coli elongation factor Tu. The hydrolysis patterns in the isolated state were similar for all aminoacylated tRNAs except Glu-tRNA2Glu, which exhibited striking differences probably arising from the existence of several subpopulations of tRNA2Glu. When engaged in a ternary complex with EF-Tu and GTP, the aminoacyl-tRNAs were efficiently protected in the amino acid acceptor and TΨC helices, showing that the interaction with EF-Tu primarily takes place at the -C-C-A end and at the amino acid acceptor and TΨC helices. In all cases an increased reactivity of the anticodon stem was observed in the complexed tRNA, possibly resulting from a conformational change in this region of the tRNAs.

Formylation of mischarged E. coli tRNA Met f

ha previous work we demonstrated the mischarging of fG&erichio coli tRNAF” by yeast phenyManyC and va&CtRNA synthetam [ 1 . The possibility of kI obtaining phenylalanyl-tRNA, ct and valy CtFWA~” allows the study of &he properties of these rnisdlarged +&of initiator t&WA durlag the different steps of ibe idtiatidn process of protehb synthesis. T&e first reaction of the initiation mechanism in JF. cofi is thf~ formy’_tton of methionyl-tRNAF”. It was therefore _=q flr3t to ve.rkfy if an incorrectly aminoacylated initiitc

Nucleotide sequences of the T1 and pancreatic ribonuclease digestion products from some large fragments of the 23S RNA of Escherichia coli.

!RNA is able to be formylated. In the present paper we demonstrate that eilher ph*oylalsnyl-tRNA~” orvalyl-tRNAys . cafl be formylaced in the presenw of the tkmsfonnylase from & coli. Thcsz results suggst that the specifkity of the formylation reaction exclusively depends upon the nature of the tRNA moiety of the aminoacylated tRNAF”’ and not upon tit of the amG10 acid bound to the tRNA.

Yeast tRNAAsp-aspartyl-tRNA synthetase: the crystalline complex.

When the 23S RNA from E. Coli was pretreated for 1 h at 60 degrees in the presence of Mg++ and K+ and then subjected to T1 ribonuclease attack, resistant fragments were recovered from 3 regions of the molecule: region A (containing 470-500 nucleotides) located at the 5 end of 23S RNA, region B (containing 520-550 nucleotides) located at the 3 end and region C (containing 110-120 nucleotides) lying between region A and region B. The nucleotide sequences of the T1 and pancreatic ribonuclease digestion products from these 3 regions have been studied and in most cases determined. In the course of these studies, a certain number of abnormal nucleotides, which are not methylated, have been encountered. A low level of sequence heterogeneity was detected.

A study of the interaction of Escherichia coli initiation factor IF2 with formylmethionyl-tRNAMetf by partial digestion with cobra venom ribonuclease

Aspartyl-tRNA synthetase from yeast, a dimer of molecular weight 125,000 and its cognate tRNA (Mr = 24,160) were co-crystallized using ammonium sulfate as precipitant agent. The presence in the crystals of both components in the two-to-one stoichiometric ratio was demonstrated by electrophoresis, biological activity assays and crystallographic data. Crystals belong to the cubic space group I432 with cell parameter of 354 A and one complex particle per asymmetric unit. The solvent content of about 78% is favorable for a low resolution structural investigation. By exchanging H2O for D2O in mother liquors, advantage can be taken from contrast variation techniques with neutron radiations. Diffraction data to 20 A resolution were measured at five different contrasts, two of them being close to the theoretical matching point of RNA and protein in the presence of ammonium sulfate. The experimental extinction of the diffracted signal was observed to be close to 36% D2O, significantly different from the predicted value of 41%. The phenomenon can be explained by the existence of a large interface region between the two tRNAs and the enzyme. These parts of the molecules are hidden from the solvent and their protons are less easily exchangeable. Accessibility studies toward chemicals of tRNAAsp in solution and in the presence of synthetase are in agreement with such a model.

Search of essential parameters for the aminoacylation of viral tRNA-like molecules. Comparison with canonical transfer RNAs.

The translation initiation factor IF-2 of prokaryotic organisms functions in the correct formation of the initiation complex fMet-tRNAfMet:mRNA:30 S ribosomal subunit ] 11. Similar to the role of the elongation factor EF-Tu in the elongation step, IF-2 can be regarded as an a~noacyl-tRNA carrier protein which ensures the correct binding of the first amino acid, formylmethionine, in the ribosomal peptidyl transferase centre. However, the binding constant of IF-2 to fMettRNA is too low to allow isolation of the complex. We have studied the interaction of IF-2 with the initiator tRNA by measuring the effect of the protein on the spontaneous hydrolysis of the aminoacyl ester bond [2]. We showed that IF-2 specifically protected the formylated form of the initiator tRNA, ~dicating that IF-2 interacts with the amino acid acceptor region of the initiator tRNA. Furthermore, we showed that this interaction was independent of GTP. To obtain more detailed information about the structural regions of the initiator tRNA involved in the binding to IF-2, we have employed the method of specific protection by IF-2 against ribonuclease digestion of jet-tRNA.