Recognition of altered E. coli formylmethionine transfer RNA by bacterial T factor

Treatment of $\text{E.}\text{coli}$ formylmethionine tRNA with sodium bisulfite produces six C → U base changes in the tRNA structure. Four of these modifications have no effect on the ability of tRNA_f^Met to be aminoacylated or formylated. Prior to bisulfite treatment, Met-tRNA_f^Met is not able to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. After bisulfite treatment, a large portion of the modified tRNA is bound as T-GTP-Met-tRNA_f^Met. Formylation of bisulfite-modified Met-tRNA_f^Met completely eliminates T factor binding. Unmodified tRNA_f^Met is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5′ terminus. Bisulfite-catalyzed conversion of this unpaired C₁ to U₁ results in formation of a normal U₁-A₇₃ base pair at the end of the acceptor stem. It is likely that this structural alteration is responsible for the recognition of bisulfite-modified Met-tRNA_f^Met by T factor.     

Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

Sequences of initiator and elongator methionine tRNAs in bean mitochondria

Summary Two bean mitochondria methionine transfer RNAs, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced usingin vitro post-labeling techniques.One of these tRNAs^Met has been identified by formylation using anE. coli enzyme as the mitochondrial tRNA_F ^Met. It displays strong structural homologies with prokaryotic and chloroplast tRNA_F ^Met sequences (70.1–83.1%) and with putative initiator tRNA_m ^Met genes described for wheat, maize andOenothera mitochondrial genomes (88.3–89.6%).The other tRNA^Met, which is the mitochondrial elongator tRNA_F ^Met, shows a high degree of sequence homology (93.3–96%& with chloroplast tRNA_m ^Met, but a weak homology (40.7%) with a sequenced maize mitochondrial putative elongator tRNA_m ^Met gene.Bean mitochondrial tRNA_F ^Met and tRNA_m ^Met were hybridized to Southern blots of the mitochondrial genomes of wheat and maize, whose maps have been recently published (15, 22), in order to locate the position of their genes.     

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA_f^Met). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA_f^Met, as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA_f^Met to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA_f^Met. This accessory factor might be unnecessary when formylated Met-tRNA_f^Met is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Δfmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA_f^Met due to loss of the MIS1 gene that encodes the mitochondrial C₁-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.     

A functional interaction between methionyl-transfer RNA hydrolase and a transfer RNA binding factor

Met-tRNA_f bound at low Mg ion concentrations to rabbit reticulocyte 40 S ribosomal subunits in the presence of ApUpG and a eukaryotic tRNA binding factor serves readily as a substrate for a Met-tRNA hydrolase from rabbit reticulocytes. This hydrolysis occurs rapidly at 0 °C, appears to be specific for Met-tRNA_f, and is not inhibited by 60 S ribosomal subunits. These reactions may be responsible for the accumulation of deacylated tRNA_f^Met observed in ribosomes isolated from sodium fluoride-treated cells.     

Highly charged radioactive initiator methionyl transfer RNA from in vivo labeled HeLa cells

³⁵S-Labeled Met-tRNA_f^Met can be prepared from HeLa cells, for studies of translation in vitro, with both a high degree of charging and a relatively high specific radioactivity. HeLa cells are labeled with [³⁵S]methionine in vivo, in the presence of cycloheximide to reduce translation. Their cytoplasmic RNA is then isolated by phenol extraction and subjected to cellulose ion-exchange chromatography in order to partially purify labeled Met-tRNA_f^Met and resolve it from Met-tRNA_m^Met.     

Incorporation of methionine from met-tRNA-Met-F into internal positions of polypeptides by mouse liver polysomes

Two methionine accepting tRNA species corresponding to tRNA_F^Met and tRNA_M^Met from mouse ascites tumor cells were tested for their ability to donate methionine into internal positions of growing polypeptide chains on mouse liver polysomes. Both tRNA species can function in the elongation of polypeptide chains as judged by their ability to incorporate methionine into protein in the absence of chain initiation. The insertion of methionine into internal positions of polypeptide chains from Met-tRNA_F^Met was confirmed by Edman degradation and CNBr cleavage. When both tRNA^Met species were present in saturating concentrations in the cell-free system a strong preference for the incorporation of methionine from Met-tRNA_M^Met became apparent.     

Isolation and characterization of two forms of Met-tRNAf deacylase from rabbit reticulocyte ribosomes

We have separated and purified two forms of Met-tRNA_f deacylase (or two separate enzymes), an activity that mediates in part the suppression of polypeptide chain initiation that occurs in heme deficiency or with double-stranded RNA, 1000-fold from the 0.5 M KCl wash of rabbit reticulocyte ribosomes. Deacylase I is a minor activity with an S_20,w of 5.9, D_20,w of 4.9 and M_r of 110 000, while deacylase II is the major activity with an S_20,w of 3.3, D_20,w of 7.1 and M_r of 43 000. Both convert crude reticulocyte or pure yeast, wheat germ, and E. coli [³⁵S]Met-tRNA_f to [³⁵S]methionine and tRNA^Met_f and have no effect on reticulocyte [³⁵S]fMet-tRNA_f, [³H]Ala-tRNA or [³H]Lys-tRNA. However, while deacylase I has similar activity throughout the pH range of 6.1–8.1, deacylase II has a sharp pH optimum at 7.9 and is almost completely inactive at 6.1. In addition, deacylase II shows a much greater affinity for pure Met-tRNA_f than deacylase I (K_m of 1.5–3 nM vs. 100 nM), and, while deacylase II is selectively inhibited by tRNA^Met_f, deacylase I is inhibited similarly by any added tRNA.     

AIMP3/p18 Controls Translational Initiation by Mediating the Delivery of Charged Initiator tRNA to Initiation Complex

Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are nonenzymatic scaffolding proteins that comprise multisynthetase complex (MSC) with nine aminoacyl-tRNA synthetases in higher eukaryotes. Among the three AIMPs, AIMP3/p18 is strongly anchored to methionyl-tRNA synthetase (MRS) in the MSC. MRS attaches methionine (Met) to initiator tRNA (tRNA_i^Met) and plays an important role in translation initiation. It is known that AIMP3 is dispatched to nucleus or nuclear membrane to induce DNA damage response or senescence; however, the role of AIMP3 in translation as a component of MSC and the meaning of its interaction with MRS are still unclear. Herein, we observed that AIMP3 specifically interacted with Met-tRNA_i^Metin vitro, while it showed little or reduced interaction with unacylated or lysine-charged tRNA_i^Met. In addition, AIMP3 discriminates Met-tRNA_i^Met from Met-charged elongator tRNA based on filter-binding assay. Pull‐down assay revealed that AIMP3 and MRS had noncompetitive interaction with eukaryotic initiation factor 2 (eIF2) γ subunit (eIF2γ), which is in charge of binding with Met-tRNA_i^Met for the delivery of Met-tRNA_i^Met to ribosome. AIMP3 recruited active eIF2γ to the MRS-AIMP3 complex, and the level of Met-tRNA_i^Met bound to eIF2 complex was reduced by AIMP3 knockdown resulting in reduced protein synthesis. All these results suggested the novel function of AIMP3 as a critical mediator of Met-tRNA_i^Met transfer from MRS to eIF2 complex for the accurate and efficient translation initiation.     

Initiation of mammalian protein synthesis: Dynamic properties of the assembly process in vitro

Binding of the Met-tRNA^Met_f·eIF-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNA^Met_f exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNA^Met_f binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNA^Met_f mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.     

Purification Of Methionine Acceptor tRNA On Arginine-Agarose

Crude E. coli tRNA or enriched methionine acceptor tRNA can be separated into three stiecies on a column of arginine-agarose. The first peak eluted is tRNA^Met and the latter two peaks are two forms of tRNA^Met _f. From crude tRNA, tRNA^Met _m is obtained in approximately 50% purity. Arginine-agarose separates enriched methionine accepting tRNA into three homogeneous fractions.     

Two forms of methionyl-transfer RNA synthetase from Mycobacterium smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from $\text{Mycobacterium smegmatis}$. The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNA_m^Met and tRNA_f^Met; enzyme B exhibited much higher specificity for tRNA_f^Met. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 10⁶ as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.     

Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast

Chemically formylated Met-tRNA_m^Met and Met-tRNA_f^Met species from $\text{E.}$$\text{coli}$ and yeast were tested for their capacity to serve as chain-initiators in a cell-free system from $\text{E.}$$\text{coli}$. In the presence of R 17 mRNA, initiation factors and $\text{E.}$$\text{coli}$ ribosomes, all four Met-tRNAs could form functional initiation complexes as measured by ribosomal binding kinetics, fMet-puromycin formation and synthesis of a dipeptide fMet-Ala. Unformylated Met-tRNA_f^Met from $\text{E.}$$\text{coli}$ displayed significantly less activity as a peptide chain-initiator than the formylated Met-tRNA_m^Met species from $\text{E.}$$\text{coli}$ and yeast. Although the latter tRNAs were less effective initiators than the “physiological” initiator tRNAs, the data seem to indicate that a blocked α-amino group represents the major token of identification by which Met-tRNA is admitted to function in $\text{E.}$$\text{coli}$ peptide chain initiation.     

Protein synthesis in rabbit reticulocytes. XIII. Lack of mRNA (poly r (A)) binding activity in highly purified EIF-1.

Met-tRNA_f^Met binding factor (EIF-1) has been purified more than 100 fold over crude high salt (0.5 M KCl) ribosomal wash. The purified factor binds 2 nmoles Met-tRNA_f^Met per mg protein and shows very little poly r(A) binding activity. Crude ribosomal high salt wash possesses significant amounts of poly r(A) binding activity and also binds to other RNAs. The bulk of this unspecific RNA binding protein is separated from EIF-1 by DEAE-cellulose chromatography.     

Transfer of methionyl residues by leucyl,phenylalanyl-tRNA-protein transferase

Leucyl, phenylalanyl-tRNA-protein transferase also catalyzes transfer of methionyl residues as indicated by (i) copurification over a 1000-fold range of transfer activities for all three amino acids and (ii) loss of methionyl transfer activity in a mutant of $\text{E.}\text{coli}$ lacking the transferase and reappearance of this activity in a transferase revertant. The purified enzyme was found to use Met-tRNA_m^Met in preference to Met-tRNA_f^Met as donor substrate. Peptides containing a basic amino acid at the NH₂-terminus functioned as acceptors for the transfer of methionyl residues.     

Association of a GDP binding activity with initiation factor eIF-2 from calf liver

A highly purified preparation of the eucaryotic initiation factor eIF-2 from calf liver which forms a ternary complex with GTP and Met-tRNA_f^Met also exhibits a potent GDP binding activity. The factor preparation specifically forms a binary complex with GDP, other ribonucleoside diphosphates and GTP are inactive. Evidence is presented indicating that the GTP-dependent Met-tRNA_f^Met binding and binary complex formation with GDP are mediated by the same protein which has an apparent molecular weight of 67,000 as judged by glycerol density gradient centrifugation.     

Interaction of rat liver ribosomal subunits with homologous Met-tRNAs

Rat liver 40 S ribosomal subunits, in the presence of magnesium ions, bind homologous, resolved Met-tRNAs in the absence of added exogenous proteins. The interaction of the aminoacyl-tRNAs with the particle is dependent on the concentration of magnesium ions in the incubation. At various Mg²⁺ concentrations examined, binding of the putative initiator Met-tRNA_i to 40 S subunits is greater than that observed with Met-tRNA_m. Also, binding of Met-tRNA_i to 40 S subunits is greater than that obtained with 40 S plus 60 S particles. The initial rate of formation of the 40 S·Met-tRNA_i complex is greater at 25 °C than at 37 or 4 °C; decay of the complex, which is observed after 15 min of incubation, is greater at 37 °C but it is slower if 60 S subunits are added after the complex has been formed. If 60 S subunits are added to the incubation with 40 S subunits at the start of the reaction, binding of Met-tRNA_i is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNA^Met) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNA_i binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNA_i and, after addition of 60 S subunits, reacts extensively with puromycin; the addition of elongation (translocation) factor EF-2 and GTP do not affect the extent of the puromycin reaction, suggesting that the acMet-tRNA_i is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

Identification of a protein at the ribosomal donor-site by affinity labeling

p-nitrophenylcarbamyl-methionyl-tRNA_f^Met is shown to act as an analogue of fMet-tRNA_f^Met in initiation complex formation. It binds to E. coli ribosomes in the presence of initiation factors and R 17-RNA as messenger. Covalent bond formation occurs in the complex between the Met-tRNA_f^Met derivative and protein of the 50 S ribosomal subunit. The protein labeled predominantly in the reaction has been identified as L 27 indicating that this protein is located at the donor-site of the ribosome.     

Crystallization of tRNAs as Cetyltrimethylammonium Salts

VARIOUS species of transfer RNA have been crystallized by controlled precipitation from aqueous solutions containing organic solvents or ammonium sulphate (reviewed in refs. 1 and 2). These methods have produced a great variety of crystal forms which, with a few exceptions^3,4, are usually poorly ordered as judged by X-ray diffraction. This is probably because the interactions between molecules are few and rather nonspecific, making the crystal structure extremely sensitive to the crystallization conditions. For this reason, attempts have been made to crystallize tRNA as the cetyltrimethylammonium (CTA-) salt. The additional interaction between hydrophobic cetyl cations bound to the different molecules may stabilize the crystal lattice and have a positive effect on the crystallization process and therefore on the order of the crystals. We report here the production of crystals of CTA-salts of five different tRNAs; tRNA^Met_f, tRNA^Glu, tRNA^Phe, tRNA^Tyr from E. coli and tRNA^Phe from yeast. In the case of tRNA^Met_f, different crystal forms were obtained in the presence of different cations.