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 tRNAIle, tRNALeu, tRNALys, tRNAMet and possibly tRNAA la. Similar comparisons of tRNAAsn, tRNAAsp, tRNAHis, tRNAPhe, tRNASer and tRNATyr have been performed as well; however, no prominent differences were observed.     

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.     

Multiple isoacceptor forms of several transfer ribonucleic acids in a mutant yeast strain.

By use of reverse phase 5 chromatography, a strain of Saccharomyces cerevisiae (XB 109-5B) has been shown to exhibit multiple isoaccepting forms for several of the transfer ribonucleic acids (tRNAs). This is in contrast with a standard wild-type strain where only one acceptor is found for each tRNA studied. Multiple peaks for tRNATyr, tRNAPhe, tRNASer, and tRNAVal have been detected for strain XB 109-5B. However, the observation of multiple isoacceptors cannot be extended to all tRNAs in this strain since tRNAAsp appears as a single form that is the same as in the wild type. The appearance of multiple peaks was found to depend on the growth conditions of the cells. The tRNA profiles of XB 109-5B that was grown rapidly with vigorous aeration differed the most from profiles of comparably grown wild-type yeast, whereas tRNA from this mutant, grown without shaking or supplementary aeration, appeared the same as the wild type. The minor nucleoside composition of the isoacceptors of tRNAPhe was obtained.     

Isoaccepting lysine transfer ribonucleic acid species of Pseudomonas aeruginosa.

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide, three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAE-Sephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNA Lys1 and a minor, tRNALys1. Co-chromatography of 14C-labelled tRNALys1 and 3H-labelled tRNALys2 on benzoylated DEAE-cellulose at pH 4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,G1) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.     

Alterations in SVT2 cell transfer RNAs in response to cell density and serum type.

The chromatographic elution profiles of tRNA-A sn, tRNA-A sp, tRNA-H is and tRNA-T y r from SV40-transformed BALB-3T3 cells grown in fetal calf serum or cald serum-supplemented media have been examined. The relative proportions of certain of the isoaccepting species of these four tRNAs are altered in a similar fashion depending on the serum type. It is suggested that the elution profile alterations reflect the extent of modifications of a specific G residue to the minor nucleoside Q, and that this process differs between untransformed and transformed cells. In addition, cell density appears to influence the Q content of these tRNAs, though other density-dependent tRNA modifications also appear to occur.     

Lysine tRNA and cell division: a G1 cell cycle mutant is temperature sensitive for the modification of tRNA5Lys to tRNA4Lys.

Ts-694 is a temperature sensitive mutant of hamster cells which is blocked in the G1 phase of the cell cycle at the restrictive temperature of 39 degrees. A comparison of the Lys-tRNA isoacceptors by RPC-5 chromatography showed a decrease in tRNA5Lys and an increase in tRNA4Lys at 39 degrees. This was identical to the changes seen in confluent cultures at the permissive temperature of 33 degrees. These Lys-tRNA changes were not seen in ts-694 cells blocked in G1 by isoleucine deficiency, nor in two other G1 ts mutants at the restrictive temperature. Cells trapped in S phase by a thymidine block also contained decreased levels of tRNA4Lys when raised to 39 degrees. Both tRNA4Lys levels and cell division increased when the cells were returned to the permissive temperature. An in vitro assay was established for the modification of tRNA5Lys to tRNA4Lys with tRNA6Lys and tRNA2Lys as intermediates. The first reaction is the synthesis of tRNA6Lys which involves the introduction of a modified uridine at the third position of the anticodon. Extracts of 694 cells grown at 33 degrees were able to modify rat liver [3H] tRNA5Lys to tRNA6Lys and tRNA4Lys in vitro when assayed at 25 degrees but not at 39 degrees. Extracts of Balb/c 3T3 cells, however, were more active at 39 degrees than at 25 degrees showing that the normal enzyme is not temperature sensitive. Ts-694 cell tRNA, isolated from cells grown at 33 degrees was aminoacylated at both 25 degrees and 39 degrees with rat liver synthetases. tRNA4Lys was present at both temperatures indicating that ts-694 cells do not contain a temperature sensitive tRNA4Lys.     

Codon-induced transfer RNA association. A property of transfer RNA involved in its adaptor function?

It is shown by equilibrium sedimentation that the binding of cognate codons to tRNAPhe (yeast), tRNAPhe (Escherichia coli), tRNALys, tRNAfMet and of the wobble codon UUU to tRNAPhe (yeast) induces dimerization of codon transfer RNA complexes. Analysis of the sedimentation profiles with a quantitative evaluation of the coupling between sedimentation and association equilibrium provides dimerization constants in the range from 1 X 10(4) to 6 X 10(4) M-1. These results on various tRNAs from different organisms suggest that the codon-induced tRNA association is a general phenomenon. Probably the codon-induced tRNA association facilitates the aminoacyl transfer reaction.     

A phase relationship associates tRNA structural gene sequences with nucleosome cores

DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.     

Interrelationships of isoacceptor phenylalanine tRNA species of Rhodopseudomonas sphaeroides.

The phenylalanine tRNA of Rhodopseudomonas sphaeroides was fractionated on benzoylated diethylaminoethyl-cellulose into four isoaccepting species (tRNAPheI to IV). tRNAPheIII represented 80% of the total tRNAPhe in anaerobic, photosynthetically grown organisms, whereas in cultures grown aerobically for prolonged periods, tRNAPheII represented 80% of the total. In cultures adapting to aerobic growth, the addition of rifampin resulted in a tRNAPhe profile characteristic of anaerobic-photosynthetic conditions due to the conversion of tRNAPheII to tRNAPheIII. In fully adapted aerobic cultures, this conversion was inhibited in the presence of chloramphenicol or rifampin. The conversion of tRNAPheIII to tRNAPheII was not observable in vivo. It is proposed that an enzymic activity synthesized during anaerobic-photosynthetic growth was responsible for the conversion.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Myeloblastosis Virus

Avian myeloblastosis virus (AMV) 4S RNA was tested for amino acid acceptor activity for 18 of the 20 amino acids. A nonrandom distribution of viral tRNAs was found compared with tRNA from normal liver or from AMV-infected leukemic myeloblasts, confirming previous reports. Methionine and proline tRNAs were considerably enriched, whereas glutamic acid, glutamine, serine, tyrosine, and valine tRNAs were markedly depleted in AMV relative to homologous cellular tRNAs. The seven AMV tRNAs with the greatest amino acid acceptance capacities, which were in order methionine, proline, lysine, arginine, histidine, isoleucine, and threonine tRNAs, were compared with homologous tRNAs from leukemic myeloblasts and liver by reversed-phase 5 chromatography. Of the 25 isoaccepting chromatographic fractions identified, no tRNA species unique to AMV was detected. Only methionyl-tRNA showed a substantial quantitative variation in isoaccepting species compared with the host cell. Thus, viral selectivity for amino acid-specific tRNAs is not, generally, paralleled by selectivity for individual isoaccepting tRNA species. Qualitative differences in arginyl- and histidyl-tRNA isoaccepting species were discovered in virus and leukemic myeloblasts compared with liver. This indicates the existence of structural differences in these tRNA species which could be related to virus replication or expression.     

Incorporation of lysine into Y base of phenylalanine tRNA in Vero cells.

Vero cells, a line derived from African green monkey kidney, contains a hypermodified base, called Y, adjacent to the 3' end of the anticodon of tRNAPhe. Two types of evidence are presented suggesting that lysine is involved in biosynthesis of Y base in these cells. First, when Vero cells are starved for lysine, a new, early-eluting species of tRNAPhe which lacks the fully modified Y base can be detected by reversed phase chromatography (RPC-5). After addition of lysine to the medium, this new species disappears. Second, when these cells are grown in low-lysine medium and then exposed to [3H]lysine, radioactivity from the lysine comigrates with tRNAPhe. The Y base can be selectively excised from tRNAPhe by incubation at pH 2.9, and extracted into ethyl acetate. Thin-layer chromatography of acid-excised material from these cells reveals that lysine-derived radioactivity comigrates with genuine Y base from calf liver tRNAPhe and the acid-excised tRNA no longer contains radioactivity. These results are consistent with the model that lysine is a structural precursor of Y base in tRNAPhe of Vero cells.     

A comparison of transfer RNA isoaccepting species between collagenous and noncollagenous tissues in the embryonic chick

Transfer RNA was isolated from different organs of 17-day-old chick embryos and the acceptor activity for each of the 20 amino acids was determined. The most abundant acceptor activities found in tRNA from tendon cells were for glycine, arginine, proline and alanine. When compared to the average acceptor activity found in brain, liver and heart, the tendon tRNA showed an increase in acceptor activity of 33% in glycine, 40% in arginine and 83% in proline. Reversed phase chromatography of the tRNA charged with glycine demonstrated that the increase in glycyl-tRNA in tendon could be accounted for by an increase in one of four major isoaccepting species. Such an increase in a single species was also observed in tRNA isolated from calvaria. The codon response of this species was shown to differ from that of the other glycyl-tRNA species. No major differences in the relative proportions of isoaccepting species could be demonstrated for any other amino acid. These results suggest that a characteristic complement of tRNA species may be associated with collagen synthesis.     

Study of yeast mitochondrial tRNAs by two-dimensional polyacrylamide gel electrophoresis: characterization of isoaccepting species and search for imported cytoplasmic tRNAs.

By two-dimensional polyacrylamide gel electrophoresis, yeast mitochondrial tRNA is fractionated into 27 major species. All but 6 of them migrate distinctly from cytoplasmic tRNAs. Migration of mitochondrial DNA-coded mitochondrial tRNAs shows the occurence of only one cytoplasmic tRNA in mitochondria. Several mitochondrial tRNA spots are identified on the electrophoregrams, some of them show isoaccepting species (Val, Ser, Met, Leu). It is suggested that there are sufficient mitochondrial tRNA genes on yeast mitochondrial DNA to allow mitochondrial protein biosynthesis by the mitochondrial tRNAs alone. Guanosine + Cytidine content and rate base composition are reported for some individual species. Mitochondrial tRNAPhe lacks Ribothymidine.     

Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae.

A mutation in Saccharomyces cerevisiae, designated mia, is responsible for the production of isoaccepting tRNA molecules with reduced extents of nucleoside modifications. The mia isoacceptors of tRNAPhe and one of the mutant isoacceptors of tRNATyr were highly purified for nucleoside composition analyses. The data indicate that the mutant isoacceptors are lacking some of the dihydrouridine moieties. This is consistent with our previous hypothesis that the mutant isoacceptors were accumulated due to a defect in a modification process [Lo, R.Y.C. and Bell, J.B. (1981) Current Genetics 3, 73-82). Data from in vitro poly-U translation experiments also support the previous results, suggesting in vivo biological activity of these mutant tRNAs.