Photoreaction of 8-methoxypsoralen with yeast-tRNAPhe. Identification of the major reaction sites

Yeast tRNAPhe was photoreacted with [3H]8-methoxypsoralen and the product was digested with ribonuclease T1, ribonuclease A or a combination of the two or cleaved with sodium borohydride/aniline. The oligonucleotides from these digestions were analyzed by polyacrylamide gel electrophoresis or high-pressure liquid chromatography and the psoralen-containing fragments were identified. The results indicate that one major and two minor photoreaction sites for 8-methoxypsoralen exist in yeast tRNAPhe. The major site (containing about 55% of the label) was determined as U50 in the T psi arm of the tRNA molecule while the minor sites were assigned to U59 (30% of the label) and C70 (15%) respectively. Our results suggest that psoralens may be used as photoprobes for studying conformational changes in tRNA molecules.     

Specific spin-labeling of transfer ribonucleic acid molecules.

The spin labels anhydride (ASL), bromoacetamide (BSL) and carbodiimide (CSL) were used to label selectively tRNAGlu, tRNA fMet and tRNAPhe from E. coli. The preparation and characterization of the sites of labeling of eight new spin-labeled tRNAs are described. The sites of labeling are: s2U using ASL, BSL and CLS and tRNAGlu; s4U using ASL and BSL on tRNAfMet and tRNAPhe; U-37 with CSL on tRNfMet; U-33 with CSL on tRNAPhe. The rare base X at position 47 of tRNAPhe has been acylated with a spin-labeled N-hydroxysuccinimide (HSL). The 3'end of unfractionated tRNA molecules has been chemically modified to a morpholino spin-labeled analogue (MSL). Their respective e.s.r. spectra are reported and discussed.     

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.     

Enzymatic conversion of guanosine 3'' adjacent to the anticodon of yeast tRNAPhe to N1-methylguanosine and the wye nucleoside: dependence on the anticodon sequence.

N1-Methylguanosine (m1G) or wye nucleoside (Y) are found 3' adjacent to the anticodon (position 37) of eukaryotic tRNAPhe. The biosynthesis of these two modified nucleosides has been investigated. The importance of the type of nucleosides in the anticodon of yeast tRNAPhe on the potentiality of this tRNA to be a substrate for the corresponding maturation enzyme has also been studied. This involved microinjection into Xenopus laevis oocytes and incubation in a yeast extract of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent Y nucleoside were substituted by various tetranucleotides ending with a guanosine. The results obtained by oocyte microinjection indicate: that all the restructured yeast tRNAsPhe are efficient substrates for the tRNA (guanosine-37 N1)methyltransferase. This means that the anticodon sequence is not critical for the tRNA recognition by this enzyme; in contrast, for Y nucleoside biosynthesis, the anticodon sequence GAA is an absolute requirement; the conversion of G-37 into Y-37 nucleoside is a multienzymatic process in which m1G-37 is the first obligatory intermediate; all the corresponding enzymes are cytoplasmic. In a crude yeast extract, restructured yeast tRNAPhe with G-37 is efficiently modified only into m1G-37; the corresponding enzyme is a S-adenosyl-L-methionine-dependent tRNA methyltransferase. The pure Escherichia coli tRNA (guanosine-37 N1) methyltransferase is unable to modify the guanosine-37 of yeast tRNAPhe.     

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.     

3'' Terminal labelling of RNA of RNA with beta-32P-pyrophosphate group and its application to the sequence analysis of 5S RNA from Streptomyces griseus.

Nucleotide pyrophosphate transferase isolated from Streptomyces griseus is used to transfer pyrophosphate group from gamma-32P-ATP to the 3'-OH of tRNA, generating a strictly terminal label at its 3' end. Using yeast tRNAPhe as model compound, it is demonstrated that the labelled molecule is suitable for rapid gel sequencing by both enzymatic and chemical methods. RNA molecules terminated by pyrimidine nucleoside are poor pyrophosphate acceptors. To label RNAs of this kind, first guanosine 5'-phosphate 3'-(beta-32P)-pyrophosphate (pGpp) is prepared from gamma-32P-ATP and GMP by nucleotide pyrophosphate transferase. pGpp is then ligated to the 3' end of RNA by T4 RNA ligase. The complete nucleotide sequence of 5S RNA from Streptomyces griseus is established by rapid gel sequencing methods performed on 3'-(beta-32P)-pyrophosphate labelled molecule.     

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.     

The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.     

Alkylation of tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of d(ATTTTCA)

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of oligonucleotide d(ATTTTCA) complementary to the sequence UGAAms2i6AA psi in the anticodon loop of tRNAPhe was studied. Three guanine residues--G28/29, G24 and G10 were found to be alkylated. Two binding sites for the reagent in the tRNA were assumed to be present. The efficiency of the alkylation of tRNA from these sites as well as an average association constant (Ka 3,8 X 10(3)M-1) for the reagent interaction with tRNA were evaluated.     

A nonspecific inhibitory effect of tRNA on the activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

The inhibitory effect of tRNA on yeast 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) has been reinvestigated. From earlier studies the inhibition by tRNAPhe appeared to be quite specific. This study shows that tRNAPhe is indeed a potent inhibitor but so is unfractionated tRNA, as well as ribosomal RNA and heparin. Complete digestion to mononucleotides relieves the inhibition. Since the enzyme requires a metal ion (Co2+) we suggest that the RNA and heparin are inhibitory by virtue of their capacity to chelate the Co2+.     

A phenylalanine tRNA gene from Neurospora crassa: conservation of secondary structure involving an intervening sequence.

We have isolated and sequenced a tRNAPhe gene from Neurospora crassa. Hybridization analyses suggest that trnaPhe is the only tRNA encoded on the cloned 5 kb DNA fragment. The tRNAPhe gene contains an intervening sequence 16 nucleotides in length located one nucleotide 3' to the anticodon position. The tRNAPhe coding region of Neurospora and yeast are 91% conserved, whereas their intervening sequences are only 50% identical. The pattern of sequence conservation is consistent with a proposed secondary structure for the tRNA precursor in which the anticodon is base paired with the middle of the intervening sequence and the splice points are located in adjacent single-stranded loops. The DNA sequence following the tRNAPhe coding region is similar to sequences following other genes transcribed by RNA polymerase III in that it is AT-rich and includes a tract of A residues in the coding strand. In contrast, the sequence preceding the Neurospora tRNAPhe coding region does not resemble sequences preceding other sequenced tRNA genes.     

Imino proton NMR assignments and ion-binding studies on Escherichia coli tRNA3Gly

The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs. The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied. Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop. The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form. The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends. Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe. 31P NMR spectra of E. coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E. coli tRNAPhe and yeast tRNAPhe. In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop. Ion binding E. coli tRNA3Gly and E. coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation.     

Enzymatic 2''-O-methylation of the wobble nucleoside of eukaryotic tRNAPhe: specificity depends on structural elements outside the anticodon loop.

We have investigated the specificity of the enzyme tRNA (wobble guanosine 2'-O-)methyltransferase which catalyses the maturation of guanosine-34 of eukaryotic tRNAPhe to the 2'-O-methyl derivative Gm-34. This study was done by micro-injection into Xenopus laevis oocytes of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent nucleotide 'Y' were substituted by various tetranucleotides. The results indicate that the enzyme is cytoplasmic; the chemical nature of the bases of the anticodon and its 3' adjacent nucleotide is not critical for the methylation of G-34; the size of the anticodon loop is however important; structural features beyond the anticodon loop are involved in the specific recognition of the tRNA by the enzyme since Escherichia coli tRNAPhe and four chimeric yeast tRNAs carrying the GAA anticodon are not substrates; unexpectedly, the 2'-O-methylation is not restricted to G-34 since C-34, U-34 and A-34 in restructured yeast tRNAPhe also became methylated. It seems probable that the tRNA (wobble guanosine 2'-O-)methyltransferase is not specific for the type of nucleotide-34 in eukaryotic tRNAPhe; however the existence in the oocyte of several methylation enzymes specific for each nucleotide-34 has not yet been ruled out.     

Reaction of tRNAPhe from yeast with 1-fluoro-2,4-dinitrobenzene. Attachment sites of the potential antigenic-determining 2,4-dinitrophenyl residues.

The reaction of 1-fluoro-2,4-dinitrobenzene with tRNAPhe from yeast, for the introduction of antigenic-determining 2,4-dinitrophenyl residues into tRNA, took place only at adenosine residues in tRNAPhe. After reaction at pH 8.0 and 50 degrees C two kinds of products were detected: one was ribose-modified adenosine which was derived from the 3' terminus of tRNA, and the other was base-modified adenosine. The sites and extent of the modification of each particular adenosine residue of tRNAPhe were determined as follows: 5 (6% modified), 31 (2%), 35 (36%), 67 (5%), and 76 (51%). Thus mainly the terminal adenosine and one adenosine in the anticodon loop bear the 2,4-dinitrophenyl residue.     

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.     

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].     

Interaction of T. thermophilus phenylalanyl-tRNA synthetase with the 3'-terminal nucleotide of tRNAPhe

The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3;-terminal nucleotide of tRNAPhe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3;-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates. tRNAPhe derivatives containing a photoreactive 4-thiouridine (tRNAPhe-s4U-76) or 6-thioguanosine residue (tRNAPhe-s6G-76) in the 3;-end have been prepared using terminal tRNA nucleotidyl transferase. Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3;-terminal adenosine in productive interaction of tRNAPhe with the enzyme: tRNAPhe-s4U-76 cannot be aminoacylated; the replacement of A-76 with s6G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased Vmax value. Relative cross-linking of the s6G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNAPhe-s4U-76. The dialdehyde derivative, tRNAPhe-Aox-76, with periodate-oxidized 3;-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNAPhe-s6G-76. The results suggest specific binding of the 3;-terminal nucleotide of tRNAPhe by the catalytic subunit of PheRS in the absence of other substrates. Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNAPhe acceptor end with PheRS. The correct positioning of the 3;-terminal nucleotide of tRNAPhe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.     

Selective alkylation of G24 residue in tRNAPhe

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of oligonucleotide d(pAACCA) was studied. G24 residue located near the sequence C17GGDA21 partially complementary to the oligonucleotide moiety of the reagent was shown to be alkylated. Oligonucleotide d(pAACCA) inhibited the alkylation. Association constant of oligonucleotide derivative with tRNAPhe (10(3) M-1) was evaluated from the dependence of the extent of tRNA modification on the concentration of the reagent. The reported method for selective alkylation of tRNA may be used for preparing photoaffinity derivatives of tRNA bearing an arylazidogroups in desired position.     

Interaction of 70 S ribosomes from Escherichia coli with spin-labeled N-Cbz-Phe-tRNAPhe. An electron paramagnetic resonance study

Two selectively spin-labeled Cbz-Phe-tRNAsPhe, one at position s4U8 and the other at position U33, have been used to study the dynamics of tRNA-ribosome interaction in the presence of poly(U) and factors washable from ribosomes. Upon binding to the ribosome, the correlation time of the spin label at position s4U8 decreases markedly while the same parameter for the label in the anticodon increases. The presence of poly(U) is not a prerequisite condition for the EPR spectral changes observed but larger variation occurs in the presence of factors washable from ribosomes. No variation in the correlation time is observed if uncharged spin-labeled tRNAPhe (on the s4U8 residue) is used in these experiments. Most of the ribosome-bound spin-labeled Cbz-Phe-tRNAPhe are puromycin-reactive, and consequently, the observed effect is manifested mainly at the ribosomal P site. These observations seem to suggest that the interaction between the N-blocked aminoacyl residue on the tRNA and the ribosome results in a conformational change on the tRNA, possibly involving tertiary interactions in a region close to s4U8. The role that the amino acid at the 3'-end can possibly play on this structural change is discussed.