tRNA Met Research Articles

Isoaccepting species differences between polysome-bound and total cellular tRNA in SVT2 cells

Reversed-phase chromatographic comparisons of total cellular tRNAs with tRNAs isolated from a polysome enriched cell fraction establish significant enrichments for specific isoaccepting species of tRNA Ile, tRNA Leu, tRNA Lys, tRNA Met and possibly tRNA Ala. Similar comparisons of tRNA Asn, tRNA Asp, tRNA His, tRNA Phe, tRNA Ser and tRNA Tyr have been performed as well; however, no prominent differences were observed.

Specific hydrolysis of methionyl-tRNA Met f catalyzed by a purified peptide.

A peptide initiation factor purified from rat liver and promoting the binding of initiator tRNA and model initiators to 40S and 80S ribosome at an acid pH liberates methionine and N-acetylmethionine from Trna Met f at neutral reaction. Phenylalanyl-tRNA, N-acetylphenylalanyl-tRNA and methionyl-tRNA Met m are not hydrolyzed under the same conditions. Hydrolysis of methionyl-tRNA Met f is stimulated by the presence of the 40S ribosomal subunit and preceeds at 37 degrees C until all the substrate has been split. No hydrolysis of initiator tRNA or N-acetylmethionyl-tRNA Met f occurs at 0 degrees C. Hydrolysis is slightly stimulated by GTP and MG2+ but not by KCl. The binding and hydrolyzing activity associated with a single protein factor may have an important function in regulating the rate of peptide initiation.

Formylation of mischarged E. coli tRNA Metf

Formylation of mischarged E. coli tRNA Metf

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 Mg 2+ 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.

Specificities of tRNA methylases from mouse liver and plasmocytoma

In vitro methylation of some Escherichia coli specific tRNAs by extracts from mouse normal liver and MOPC 173 plasmocytoma has been studied. The determination of the overall extent of methylation as well as of the repartition of methylated products reveals that tRNA methylases from these two sources have distinct specificities. In particular, plasmocytoma extracts seem to contain one methylase that does not exist in liver extract, the activity of which appears when tRNA Met f is used as a substrate.

Incorrect aminoacylations catalysed by E. coli valyl-tRNA synthetase

Summary It is possible to produce incorrect aminoacylations on yeast or E. coli tRNA's by purified valyl-tRNA synthetase from E. coli when special experimental conditions are used. These conditions differ from the classical ones by a high [Mg ++ ]/[ATP] ratio, a more alkaline pH, a long reaction time, a high enzyme concentration and in some cases the addition of dimethylsulfoxide. Under these conditions valine can be charged by E. coli valyl-tRNA synthetase on several yeast tRNA's (tRNA Val , tRNA Thr , tRNA Ile , tRNA Phe , tRNA Ala and tRNA Met ) and on a few E. coli tRNA's (tRNA Val , tRNA Ala and tRNA Met ). Some of these incorrect aminoacylation reactions have been studied more carefully using purified yeast tRNA Ala and tRNA Phe . It has been shown that the K m 's of these reactions are only increased by a factor of 10, as compared to the value obtained in the normal aminoacylation of E. coli tRNA Val , whereas the V max 's decrease strongly by a factor of 1000. It has been observed that whatever the aminoacylation conditions used, only a fraction of purified tRNA can be charged with the incorrect aminoacid. The incomplete aminoacylation is not due to enzyme inactivation, as the reaction resumes if a new amount of tRNA is added. The incorrect aminoacylations can be related to structural changes either at the enzyme or at the tRNA level. As they are found only with a limited number of tRNA's it could be that they involve some structural analogies between the tRNA normally recognised by E. coli valyl-tRNA synthetase and those tRNA's which are abnormally recognised. Sequence analogies between tRNA's of known sequence, aminoacylated without dimethylsulfoxide, are found especially in the dihydrouracil stem, in some nucleotides of the dihydrouracil loop, in the terminal part of the aminoacid acceptor stem and in the extra-loop. Some of these structural analogies are not conserved when the sequences of the tRNA's aminoacylated in the presence of dimethylsulfoxide are compared, but the similarities at the level of the extra-loop are still found.

Occurrence of cysteinyl transfer ribonucleic acid with a blocked α-amino group in rabbit reticulocytes

1. Rabbit reticulocytes or reticulocyte extracts incorporate cysteine into cysteinyl transfer ribonucleic acid (Cys-tRNA) having a blocked α-amino group (X-Cys-rRNA). The lack of a free α-amino group is revealed in the electrophoretic characteristics of X-cysteinyladenosine (X-Cys-A) obtained from X-Cys-tRNA by treatment with pancreatic ribonuclease, and in the failure of the X-Cys-A to react with acetic anhydride. The electrophoretic characteristics of X-Cys-A and its reactivity with diazomethane indicate that the blocking group X might have a free carboxyl group. X-Cys-A does not react with iodoacetate, possibly indicating that its sulfhydryl group is blocked. 2. Both high and low molecular weight fractions of the reticulocyte extract (obtained by fractionation by gel filtration through a Sephadex G-25 column) are needed for converting Cys-tRNA into X-Cys-tRNA. X-Cys-tRNA was not found in Escherichia coli extracts, but E. coli Cys-tRNA is converted to X-Cys-tRNA when added to a reticulocyte extract. 3. NaF is thought to inhibit a step in peptide chain initiation and to lead to the accumulation of initiation complexes ( W. Hoerz and K. S. McCarthy, Proc. Natl. Acad. Sci. U.S., 63 (1969) 1206). A tRNA species involved in chain initiation in reticulocytes is tRNA Met f. Intact reticulocytes treated with NaF contain about 7 times more X-Cys-tRNA and 4 times more tRNA Met f (at least partially acylated with methionine) bound to ribosomes than control cells. The amount bound to ribosomes of several other aminoacyl-tRNA's tested (including Cys-tRNA), however, decreases upon treatment with NaF. 4. The structure, biosynthesis, and function of X-Cys-tRNA remain to be established.

Differences de specificite entre les methylases de myelome et de foie vis-a-vis du tRNA d' E. coli K 12

This paper describes an attempt to find a difference between the patterns of methylation of E. coli tRNA by extracts of two mouse tissues. Two samples of tRNAs, methylated in two separate experiments with extracts of myeloma and of liver in presence of either 14C or 3H S-Adenosyl-L-Methionine, were pooled and fractionated together on a RPC column. The results show a difference in the specificities of the two extracts. Chromatography on DEAE Sephadex suggests that the tRNA Met is methylated by the enzymes on the myeloma, while enzymes from liver react very little, if at all, with that particular tRNA species. Studies have been undertaken in order to find out whether similar differences can also be demonstrated in homologous systems.

Characteristic behavior of 4-thiouridine region of individual amino acid-specific Escherichia coli tRNA's upon heat denaturation

With the use of ultraviolet absorbance at around 335 mμ, which is specific for 4-thiouracil residue in native Escherichia coli tRNA, the behavior of the 4-thiouridine region in response to heat denaturation has been investigated in comparison with that of the whole region by using seven individual amino acid-specific tRNA's. The data indicated that the melting out temperature of the 4-thiouridine region depended upon the amino acid-specific tRNA species used. In addition, the 4-thiouridine region in many of the tRNA species melted out in bi-modal fashion. Further study of the outstanding bi-modal melting of the 4-thiouridine region in tRNA Met F has indicated that a local rearrangement in the tRNA molecule occurred at around 70°, which was between the two phases. The discrepancy between the bi-modal behavior of 4-thiouridine and the fact that only one 4-thiouridine residue per tRNA molecule is present could be interpreted to suggest that the two-dimensional structure of tRNA was further folded to construct a more complicated form.