Met-tRNA Met Research Articles

To initiate or not to initiate: A critical assessment of eIF2A, eIF2D, and MCT-1·DENR to deliver initiator tRNA to ribosomes.

Selection of the correct start codon is critical for high-fidelity protein synthesis. In eukaryotes, this is typically governed by a multitude of initiation factors (eIFs), including eIF2·GTP that directly delivers the initiator tRNA (Met-tRNAi Met ) to the P site of the ribosome. However, numerous reports, some dating back to the early 1970s, have described other initiation factors having high affinity for the initiator tRNA and the ability of delivering it to the ribosome, which has provided a foundation for further work demonstrating non-canonical initiation mechanisms using alternative initiation factors. Here we provide a critical analysis of current understanding of eIF2A, eIF2D, and the MCT-1·DENR dimer, the evidence surrounding their ability to initiate translation, their implications in human disease, and lay out important key questions for the field. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Mechanisms Translation > Regulation.

The non-enzymatic specific aminoacylation of transfer RNA at high pressure

This paper shows that the phenylalanine-specific tRNA of Escherichia coli as well as the yellow lupin methionine initiator tRNA Met can be charge specifically with phenylalanine and methionine, respectively, in the absence of specific aminoacyl-tRNA synthetases, under high pressure of a maximum of 6 kbar (1 bar = 10 5 Pa; 1atm = 1.01 × 10 5 Pa). The esterification reaction takes places at the 3′ end of the tRNA molecules. The yield of Phe-tRNA Phe or Met-tRNA Met at high pressure is ∼10 times lower than that of the enzymatic aminoacylation reaction. This reaction seems to be specific, and mis-aminoacylation of tRNA Phe and tRNA Met with serine is negligible. It is well known that tRNA undergoes conformational changes during interaction with an aminoacyl-tRNA synthetase. Similarly, on the basis of circular dichroism spectra, we showed that the conformation of tRNA at high pressure differs slightly from its original A-RNA form. Therefore, it can be speculated that the chargeable conformation of tRNA induced by the aminoacyl-tRNA synthetase during enzymatic aminoacylation and the one created at high pressure are similar and are most probably formed by a dehydration mechanism. We think that the ‘unique’ tertiary structure of tRNA existing under high pressure creates an active centre which might itself catalyse ester bond formation. Therefore, the structure of the amino acid stem of tRNA may determine (code) the charging of the particular amino acid to specific tRNA. This code is clearly distinct from the rules of the classical genetic code.

Structural arrangement of the codon-anticodon interaction area in human placenta ribosomes. Affinity labelling of the 40S subunits by derivatives of oligoribonucleotides containing the AUG condon

Using the derivatives of the oligoribonucleotides pAUGU n and AUGU n C ( n = 0; 3) bearing an alkylating group at either the 5′ or 3′ end, respectively (mRNA analogues), the affinity labelling of the human placenta 40S ribosomal subunits has been investigated in model initiation complexes obtained in the presence of the ternary complex eIF-2 · GTP · Met-tRNA Met f. The regions of 18S rRNA and ribosomal proteins labelled with these mRNA analogues were identified. The sites of covalent attachment of the pAUGU n derivatives with a reactive group at the 5′ end were located between 18S rRNA positions 976 and 1164. The derivative of AUGU 3C with an alkylating group at the 3′ end modified 18S rRNA mainly at the 593–673 region. The main targets of the 3′ end derivative of AUGC were located between positions 1610 and 1869. The proteins S3/S3a, S6, S7 and S14/S15 were modified by both types of the oligoribonucleotide derivatives regardless of the point of the reactive group attachment to the oligonucleotide moiety. The proteins S2 and S4 were modified by both the 3′ end derivative of AUGC and 5′ end derivative of pAUGU 3; and the protein S8 was modified by the 3′ end derivative of AUGC. The proteins S5 and S9 were labelled by the 5′ end derivative of pAUGU 3, and the protein S17 was modified by the 5′ end derivative of pAUG.

Protection against non-enzymatic deacylation of Met-tRNA Metm by the ribosome : an approach to the mcasurement of aminoacyl-tRNA : ribosome affinity constants

When bound to E. coli 70S ribosomes in the presence of poly (A, U, G) at 37 degrees C, Met-tRNA Met m is hydrolysed 20 times slower than when free in solution under the same conditions (lifetimes of 8.3 h and 22 min respectively). It is shown how this large difference in rate of hydrolysis may be used to study the equilibrium of aminoacyl-tRNA binding to ribosomes.

The efficiency of methionine incorporation from isoaccepting species of tRNA Met into rabbit globin in an homologous reticulocyte lysate system

Rabbit globin α and β chains were labeled with [ 3H]leucine, and with [ 35S]methionine from reticulocyte tRNA Met isoacceptors using a rabbit reticulocyte cell-free synthesis system. [ 35S]Methionine from the three tRNA Met species isolated by RPC-5 chromatography was incorporated into internal positions of both α and β globin. The initiator tRNA, tRNA Met I, exhibited very low efficiency for incorporating methionine internally, while tRNA Met II was four times more efficient than tRNA Met III. Amino acid analysis of the tryptic peptides of the labeled globins revealed that all three isoacceptors incorporated methionine into the normal methionine peptides. Similar studies with Escherichia coli [ 35S]Met-tRNA Met f showed a 3-fold increase over the reticulocyte initiator tRNA in its capacity to incorporate methionine into the internal positions of rabbit globin.

FMet-tRNA Metf Binding and peptidyl transferase function in free and bound ribosomes from normal and puromycin aminonucleoside-treated rats

Treatment of rats with the aminonucleoside of puromycin, which increases the incorporation of labelled phenylalanyl-tRNA into polypeptide chains in liver ribosome preparations studied vitro, did not change the factor-dependent binding of f MettRNA Met f to ribosomes nor the peptidyl transferase function of the ribosomes. Peptidyl transferase function, as measured by fMet-tRNA Met f-puromycin formation, was comparable in the free and bound ribosome preparations. Similarly, the factor-dependent binding of fMet-tRNA Met f to ribosomes was the same in free ribosome preparations obtained from rat liver as it was in bound ribosome preparations that had been freed of membranes by puromycin incubation and high salt wash.

The ternary complex of initiation factor IF-I, Met-tRNA Metf and GTP An aurintricarboxylate-sensitive intermediate in the initiation of eukaryotic protein synthesis

We have previously described an initiation factor, extensively purified from 0.5 M KCl extracts of rabbit reticulocyte polysomes, which forms a ternary 1:1 complex with the initiator tRNA [Met-tRNA Met f (rabbit liver)] and GTP. We now refer to this initiation factor as IF-I. When the ternary complex was separated from GTP and [ 35S]Met-tRNA Met f by Sephadex G-100 gel filtration and used as the only source of radioactive methionine in a rabbit reticulocyte in vitro amino acid incorporation system, the transfer of [ 35S]methionine into acid-insoluble material, using A-U-G-(U) x (x = 25 ± 5) as the messenger, was greatly stimulated as compared to the transfer of [ 35S]methionine from free [ 35S]Met-tRNA Met f. Kinetic experiments now confirm that the methionine residue in the ternary complex is a preferred precursor to the N-terminal position of newly initiated polypeptide chains. The formation of the complex is inhibited by low levels of aurintricarboxylate; however, the preformed ternary complex is stable and is not dissociated by the addition of aurintricarboxylate. When the preformed ternary complex is used in the in vitro amino acid incorporation system, aurintricarboxylate inhibits the formation of acid-insoluble material, but only at levels higher than those required for the prevention of ternary complex formation. Thus one reaction in protein biosynthesis which is very sensitive to the presence of aurintricarboxylate is the formation of the ternary complex. This phenomenon may partially explain the action of aurintricarboxylate upon eukaryotic protein synthesis initiation mediated by Met-tRNA Met f.

Recognition of eukaryotic initiator tRNA by an initiation factor and the transfer of the methionine moiety into peptide linkage

A protein factor, obtained from 0.5 M KCl extracts of rabbit reticulocyte ribosomes, forms a GTP-dependent complex with the initiator tRNA from rabbit liver, Met-tRNA Met f. This activity has been purified approx. 125-fold by ion-exchange chromatography upon DEAE-cellulose and hydroxylapatite. Complex formation can be observed by virtue of the complex's elution position from Sephadex G-100 and by its retention on Millipore (nitrocellulose) filters. Complex formation is dependent on the presence of GTP; Met-tRNA Met f and GTP bind to the purified factor in a 1 : 1 ratio. 5′-Guanylylmethylenediphosphonate ( Guo-5′-P 2 CH 2P ) is 50 % as efficient as GTP, while GDP is unable to substitute for GTP. In the presence of saturating amounts of protein and GTP, complex formation is proportional to the amount of Met-tRNA Met f added. With excess amounts of GTP and Met-tRNA Met f, complex formation is proportional to the protein concentration. Heating a solution of the protein at 75 °C for 15 min destroys 80 % of the Met-tRNA Met f binding capacity. Met-tRNA Met m and Phe-tRNA Phe bind poorly to the protein. The binding of the initiator tRNA to the 40-S ribosomal subunit and to the 80-S ribosome of reticulocytes in the presence of the triplet ApUpG is dependent on the initiation factor. The factor stimulates the in vitro incorporation of methionine from [ 35S]Met-tRNA Met f into acid-insoluble material using ApUpG(pU) n ( n = 25 ± 5 ) as the messenger. The ternary complex, consisting of [ 35S]Met-tRNA Met f, GTP and factor, was separated from free GTP and [ 35S]Met-tRNA Met f by Sephad ex G-100 gel filtration. When this complex is used as the only source of radioactive methionine in an in vitro amino acid incorporation system, the transfer of [ 35S]methionine into acid-insoluble material using ApUpG(pU) n as the messenger is greatly stimulated as compared to the transfer of methionine from free [ 35S]Met-tRNA Met f or free [ 35S]-methionine.

Function of Met-tRNA fMet and Met-tRNA Met in peptide-chain elongation in cell-free systems from mouse-liver and ascites-tumor cells.

The two methionine‐accepting tRNA species from mouse ascites tumor cells were investigated with respect to their function in peptide chain elongation, using a mouse liver polysome system and a T‐factor dependent ribosomal‐binding assay. In support of results published previously it was found that in a non‐initiating cell‐free system Met‐tRNAMetf inserts methionine into internal positions of nascent peptide chains, even in the presence of Met‐tRNAMet.Correspondingly, T factor from ascites tumor cells stimulated the binding of both methionine‐accepting tRNAs to washed ribosomes. When Met‐tRNAMet and Met‐tRNAMetf were introduced together into the ribosomal binding assay, a strong preference for the T‐factor‐induced attachment of Met‐tRNAMet became visible. Formylated initiator tRNA was not recognized by T factor Whereas the ribosomal binding of formylated or unformylated initiator tRNA promoted by initiation factors functioned equally well with poly(A,U,G) and the triplet ApUpG, the T‐factor‐induced attachment of both Met‐tRNAMet species required the presence of the polynucleotide.Competition experiments with high concentration of Val‐tRNAVal and Met‐tRNAMetf revealed the occurrence of a partial mutual interference of these two tRNA species during chain elongation.The magnitude of this effect, however, was not sufficient to attribute the function of Met‐tRNAMetf in chain elongation to miscoding. No competition was observed between Val‐tRNAVal and Met‐tRNAMet.

Studies on the initiation of protein synthesis in animal tissues

Two methionine accepting tRNAs have been separated from rabbit liver tRNA by column chromatography on BD-cellulose. One of the species (Met-tRNA F Met) can be formylated using an Escherichia coli transformylase preparation, and maximal binding of this species to brain ribosomes is achieved in the presence of AUG, GTP and a 0.5 M NH 4Cl extract of the ribosomes. Transferase I cannot replace the NH 4Cl extract in the binding assay. The other tRNA species (Met-tRNA Met) requires GTP and transferase I for binding to ribosomes.