Amino acid activation, sRNA

Jhe synthesis of proteins, as characterized by the in vitro incorporation of amino acids into the protein component of cytoplasmic ribonu-cleoprotein, is known to require the nonparticulate portion of the cytoplasm, ATP (adenosine triphosphate) and GTP (guanosine triphosphate) (15, 23). The initial reactions involve the carboxyl activation of amino acids in the presence of amino acid-activating enzymes (aminoacyl sRNA synthetases) and ATP, to form enzyme-bound aminoacyl adenylates and the enzymatic transfer of the aminoacyl moiety from aminoacyl adenylates to soluble ribonucleic acid (sRNA) which results in the formation of specific RNA-amino acid complexes—see, for example, reviews by Hoagland (12) and Berg (1). The subsequent steps in pro-... 

Experimentally, C14-aminoacyl sRNA was incubated with rat liver microsomes or ribosomes, GTP, various fractions obtained from the nonparticulate portion of rat liver homogenates, and buffered salt-sucrose medium in a total volume of approximately 2 ml. (6-10). The C14-aminoacyl sRNA was prepared by the phenol-extraction procedure from the pH 5 amino acid-activating enzymes, fraction of rat liver after incubation with C14-L-amino acids (9, 13). C14-leucyl sRNA (approximately 1000 c.p.m.), having a specific radioactivity of approximately 55,000 c.p.m. per mg. of RNA, and containing a complement of endogenous, unlabeled, bound amino acids, was used in most of these studies. The microsomes were sedimented from the post-mitochondrial supernatant at 104,000 x g (10) and the ribosomes were prepared from them by extraction with deoxycholate (16). 

Previous studies by Hoagland et al. (13), Zamecnik et al. (24), and in this laboratory (9, 10) demonstrated that the transfer of amino acid from isolated sRNA-amino acid to microsomes required GTP, ATP, an ATP-generating system, and a soluble portion of the cell. Most of the aminoacyl-transferring activity present in the homogenate supernatant was recovered in the pH 5 Supernatant obtained after precipitation of the amino acid-activating enzymes at pH 5. A protein fraction, 500- to... 

The transfer of labeled amino acids from aminoacyl sRNA to purified rat-liver ribonucleoprotein particles has been shown to require GTP, and a soluble portion (pH 5 Supernatant) of the cell. An enzyme fraction, aminoacyl transferase (or polymerase) I, purified from the pH 5 Supernatant was found to catalyze the transfer of amino acid to protein with microsomes, but not with the more purified ribonucleoprotein particles (ribosomes). When transferase I was supplemented with glutathione and a microsomal extract, microsomal aminoacyl transferase (or polymerase) H, transferring activity was restored. Since the pH 5 Supernatant was active in catalyzing the transfer of amino acids from sRNA to ribosomal protein, it was concluded that both transferring activities were present in this crude fraction. Resolution of the two activities from the pH 5 Supernatant fraction was obtained by salt-fractionation procedures. Neither enzyme fraction was active when incubated individually or with glutathione, but together in the presence of... 

Chloramphenicol has been reported to have no effect on amino acid activation or incorporation of amino acids into sRNA. In high concentration, however, it inhibits nearly completely the incorporation of labelled amino acid into the protein of calf thymus nuclei . The antibiotic thus appears to interfere at a late stage in the process of protein synthesis. It is of interest that its L-erythro isomer has bttle effect on protein synthesis but inhibits the synthesis of a D-glutamyl pol5q>eptide by Bacillus suhtilis. ... 

The soluble fraction, or the pH 5 enzymes, contain amino acid activating enzymes, transfer RNA, the enzymes responsible for the terminal addition of nucleotides to the sRNA (and hence for conditioning the latter for accepting amino acids from the activating enzymes) and, probably, enzymes responsible for the transfer of the activated amino acid from the sRNA to the RNP particles. In addition, this fraction contains large amounts of unidentified protein material. 

Instead of whole supernatant or pH 5 fraction it is also possible to use isolated sRNA, either with the supernatant of the pH 5 precipitate (which contains some activating enzymes and most of the enzymes responsible for end group addition and for transferring amino acid from sRNA to particles), or with unwashed particles (which seem to have carried down enough of the necessary enzymes). Generally, the addition of isolated... 

Let us consider the first question. Since it has been shown that amino acid-RNA compounds can be formed by highly purified amino acid activating enzymes, and the properties of this reaction and of the reaction product correspond closely to those observed in crude systems or in vivo, it seems to be established beyond reasonable doubt that the mechanism of this reaction is as outlined in Section III, B, 3, d. Furthermore, the demonstration that the amino acid bound to transfer RNA can be transferred to the microsomes and be bound there in the interior of a peptide chain, seems to show that the RNA-amino acid compound can serve as a donor of amino acid for the incorporation reaction however, since not only GTP, but also ATP and soluble fraction (which mi t contribute a large number of other factors besides the transferring enzyme) are required for the transfer, the role of sRNA-amino acid is less clear-cut than it might be. A reversal of the reaction back to the adenylates, however, and incorporation by some other route seems to be excluded by the fact that even a hundredfold excess of nonisotopic amino acid does not interfere with the efficiency of the transfer, which under the right conditions, approaches 100% (14S). The evidence to date, then, indicates that the adenylate-sRNA pathway is a pathway of amino acid incorporation in the microsomal system of mammalian origin. 

Fig. 15. Over-all scbematio representation of the reactions involved in the adenylate-sRNA pathway of amino acid incorporation. Ei represents the amino acid activating enzymes E, the transferring enzymes and Es and E4, the enzymes responsible for the terminal incorporation into sRNA of ATP and CTP, respectively.

The previously observed trend of studying certain isolated components of the amino acid incorporatir stem without keeping in mind the interaction of these components in the whole system has continued, so that by far the largest number of publications in the field of protein syntheras are not really concerned with the formation of protein, or even with the incorporation of amino acids into protein, but with the study of amino acid activating enzymes, sRNA, and of course, ribonucleoprotein particles. 

Table n) in the process of purification of the transferring factor, has suggested that this enzyme may catalyze the transfer of several or perhaps all of the sRNA-bound amino acids to microsomal proteins (10). A similar suggestion is based on the fact that the transferring activity toward several amino acids is eluted in a single peak on chromatography on DEAE-cellulose (20). 

Since pH 5 Supernatant was active in the transfer of sRNA-bound amino acids, it suggested that both of the essential fractions described above were also present in this crude soluble preparation. Experimental verification of this suggestion is presented in Table V (8). Fractionation of the pH 5 Supernatant with ammonium sulfate yielded two fractions, 0 to 35% A.S. residue and 35 to 60% A.S. residue, which by themselves catalyzed aminoacyl transfer in the presence of glutathione. Reprecipitation of the 0 to 35% A.S. fraction, as described in Figure 1,... 

Amino acids are also involved in protein synthesis. Before they can be utilised, however, they must first be activated by aminoacyl-sRNA synthetases. These are specific for individual amino acids, and those so far identified are responsible for the activation of almost all the important amino acids. 

At least three stages are now believed to be involved in protein synthesis in animal cells. In stage (j), amino acids (AA) are activated by the formation of amino acyl adenylates in the presence of appropriate activating enzymes (E). In stage (2), the activated amino acid reacts with ribonucleic acids of relatively low molecular weight, known as soluble (or transfer) ribonucleic acids (sRNA). In stage (j), the amino acid is transferred to the RNA of the microsomes (Ms) where it is incorporated into new protein ... 

If we try to keep track of an amino acid on its way to protein, we will, as we have seen in the preceding section, at one point be able to recover it in close association with a particular fraction of rather low-molecular weight RNA. This particular kind of RNA, because it is not sedimented by prolonged centrifugation at 100,000 g, that is, remains soluble, has been operationally named soluble RNA or cell-sap RNA —commonly abbreviated to sRNA (18S) and because its function seems to be to accept amino acids from the activating enzymes, and to transfer them to the ribosomes, it is also desdgnated as transfer RNA (118) or acceptor RNA. ... 

The presence of biochemically active sRNA can be ascertained in several ways. The cells may be labeled with a radioactive amino acid, in vivo, for short periods of time, then fractionated, the RNA isolated and its specific activity determined. (A rough estimation of the amount of... 

Since, as we have seen, all active RNA molecules have the same terminid sequence, pCpCpA, and tince the attachment of the amino acid occurs on the terminal adenosine, the specificity of the RNA molecules must reside in the rest of the chain. Whatever this specificity contists in, however, it does not seem to be species specific, since the activating enzymes from one source can transfer amino acids specifically to RNA s of a very different origin. Hecht and co-workers (188), for example, tiiowed that the soluble fraction from mouse ascites tumor cells could transfer amino acids to RNA prepared from rat liver, calf liver, and yeast as well as to its own RNA. The soluble enzymes from guinea pig liver can transfer amino acids to RNA from rabbit (128) and from E. coli (182), although with the latter the transfer occurs at a slower rate. Furthermore, the activating enzymes and sRNA from liver and Tetrahymena (148), and from liver and spleen (69) have also been used interchangeably. 

The in vitro rate of amino acid incorporation into sRNA depends, of course, on the concentration of the activating enzymes (although these have no effect on the total amount incorporated). When one attempts, however, to view this reaction as a step in the over-all incorporation re-... 

The inhibition of amino acid incorporation into RNA by pyrophosphate and hydroxylamine are readily understood, since these are inhibitors of the activation reaction. In addition to reversing the latter, however, pyrophosphate, in crude systems, also inhibits by reversing the incorporation of terminal ATP and CTP into sRNA, hence destroying the amino acid acceptor 214). 

From the scheme, we see immediately that the presence of sRNA is essential, since here we have a RNA molecule in actual chemical combination with an activated amino acid, presumably on its way to protein. Furthermore, as we have seen, the formation of the RNA-amino acid compound is extremely sensitive to ribonuclease. In principle, therefore, this step could account for the fact that protein synthesis does not take place in the absence of RNA, and that it is inhibited by ribonuclease. Nevertheless, all evidence to date strongly indicates that the cytoplasmic... 

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