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In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryl-tRNA synthetase, and its expression in Escherichia coli. Two human brain eDNA clones of different origin, containing overlapping fragments coding for human seryl-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone). For both clones the 5′ region of the cDNA was missing. This 5′ region was obtained via PCR methods using a human brain 5′ RACE-Ready cDNA library. The complete cDNA sequence allowed us to define primers to isolate and characterize the intron/ exon structure of the serS gene, consisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp. The availability of the gone structure of the human enzyme could help to clarify some aspects of the molecular evolution of class-II aminoacyl-tRNA synthetases. The human seryl-tRNA synthetase has been expressed in E. coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA. The human seryl-tRNA synthetase sequence (514 amino acid residues) shows significant sequence identity with seryl-tRNA synthetases from E. coli (25 %), Saccharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorhabditis elegans (60%). The partial sequences from published mammalian seryl-tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl-tRNA synthetase, respectively). Human seryl-tRNA synthetase, similar to several other class-I and class-II human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal domain and a N-terminal insertion identified in the human enzyme. In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E. coli tRNA. [ABSTRACT FROM AUTHOR]

Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF-2-dependent binding of fMet-tRNA to 30-S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230–234]. The N-formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template. This paper shows that N-acetylated Phe-tRNAPhe (AcPhe-tRNA), but not Phe-tRNAPhe or tRNAPhe, competes with fMet-tRNA for binding to poly(U)-programmed 30-S ribosomal subunits. The two species of N-blocked aminoacyl-tRNA are bound to poly(U)-programmed and poly(X)-programmed 30-S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMet-tRNA or AcPhe-tRNA, whereas with poly(X) there is a 2–3-fold preference for fMet-tRNA. The initiation factor IF-2, which is strictly required for the binding of N-blocked aminoacyi-tRNAs, has a higher affinity for fMet-tRNA than for AcPhe-tRNA. It is concluded that (a) interaction of the 30-S ribosomal subunit with poly(U) or poly(X) leads to IF-2-dependent binding of N-blocked aminoacyl-tRNA; (b) the initiation factor IF-2 discriminates in favour of fMet-tRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N-blocked aminoacyl-tRNA. [ABSTRACT FROM AUTHOR]

Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenylalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The α and β subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl-tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant a subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II ts. Although the apparent affinity of tRNA for site U remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed. [ABSTRACT FROM AUTHOR]

In this paper we report on the properties of thc purified isoleucyl-tRNA synthetase from Bacillus stearothermophilus. The enzyme which was purified approximately 500-fold, has a molecular weight of about 110 000. The effect of temperature on the activation and transfer activities has been studied. The thermostable enzyme shows an optimal activity at 65 °C for the isoleucine-dependent ATP-PPi exchange reaction and at 45 °C for the formation of isoleucyl-tRNA. Attempts to heat inactivate the amino-acid-transfer activity selectively, i.e. without loss of the activation reaction, were unsuccessful. The protection of the enzyme against heat inactivation in presence of tRNA makes it possible to study the binding of this molecule to the protein. An association constant of 0.11 nM-1 was determined at 70.5 °C. Formation of this tRNA · enzyme complex was also demonstrated by binding onto nitrocellulose filters. By this technique one tRNA site per enzyme molecule was detected and an affinity constant of 0.4 nM-1 measured. The same results were obtained either with tRNA from Escherichia coli or B. stearothermophilus, showing close resemblance of the two nucleic acids. Periodate-oxidation of the 3'-terminal adenosine of the tRNA does not perceptibly affect the binding or the degree of protection of the enzyme against heat inactivation. These effects are still strictly dependent on magnesium ions and a clearcut requirement of these ions for the formation and/or stabilization of the tRNA · enzyme complex has been demonstrated. The intermediate isoleucine · AMP · enzyme complex with a molar ratio of 1:1:1 was isolated by molecular sieving and by chromatography on DEAE-cellulose. Mg2+ ions are required for the formation of the complex but are not necessary to stabilize the isoleucyl-adenylate once this is bound to the enzyme. The transfer of activated isoleucine to tRNA again requires magnesium ions and must probably be related to the formation of a tRNA enzyme complex. Part only of the activated isoleucine is transferred to tRNA; this results essentially from the breakdown of a fraction of the isoleucine · AMP · enzyme complex, induced by tRNA and Mg2+ ions, acting together. Periodate-oxidized tRNA of B. stearothermophilus or of E. coli in presence of Mg2+ ions completely releases isoleucine from the complex. [ABSTRACT FROM AUTHOR]

The binding of purified Gly3-Phe-tRNA to Escherichia coli ribosomes was compared to that of crude Gly3-Phe-tRNA (containing bulk tRNA). Gly3-phe-tRNA from the purified preparation binds preferentially to the P site (peptidyl site) at both a high (30 mM) and a low (10 mM) magnesium acetate concentration, while peptidyl-tRNA from the crude preparation bound more to A site (aminoacyl site) than to the P site at 30 mM magnesium acetate under the same experimental conditions. The addition of tRNAPhe to the purified Gly3-Phe-tRNA preparation strongly inhibited the binding at 10 mM. At 30 mM Mg2+ the addition of the same amount of tRNAPhe caused the Gly3-Phe-tRNA to bind partially to the A site, but did not inhibit the total amount of Gly3-Phe-tRNA which binds to the ribosomes. The binding of purified Phe-tRNA in the presence of T factor and GTP is not inhibited by tetracycline. However, tetracycline strongly inhibited the enzymatic binding of Phe-tRNA when tRNAPhe was added. From the results presented in this paper it is proposed that in the presence of poly(U), Phe-tRNA or peptidyl-tRNAPhe can bind to A sites only of those ribosomes which have their P site occupied by tRNA or one of its derivatives (aminoacyl-tRNA or peptidyl-tRNA). [ABSTRACT FROM AUTHOR]

Multiple species of Escherichia coli transfer RNA for valine and leucine were separated from each other by countercurrent distribution. Each transfer RNA species was charged with the corresponding radioactive amino acid and then allowed to transfer its label into hemoglobin peptides in the ribosomal system from rabbit reticulocytes. Earlier experiments had shown that the minor tRNAIIbLeu would only label one position, no. 48 of the α-hemoglobin chain under these conditions. In this paper we report the coding properties of the other separated leucine tRNAs and of multiple valine tRNAs with the hemoglobin messenger. The leucine codons in the α-chain messenger can be divided into three classes with respect to their tRNA response : (a) position 48 is recognized only by tRNAIIbLeu. (b) The majority of the leucine codons can be recognized by both the two major leucine tRNAs. (c) Some leucine codons can only be recognized by one of the major leucine tRNAs. Similarly, two classes of valine codons can be distinguished in the α-chain messenger. [ABSTRACT FROM AUTHOR]

The behavior of N-acetylphenylalanyl-tRNA as an initiator of phenylalanine polymerisation in an Escherichia coli cell-free system shows some analogy with the behavior of formylmethionyl- tRNA during the synthesis of natural proteins. At low Mg++ concentrations, polyphenylalanine chain initiation depends on N-acetylphenylalanyl-tRNA and the same initiation factors which are required for protein chain initiation with formylmethionyl-tRNA. However in a rabbit reticulocyte cell-free system, N-acetylphenylalanine is not incorporated into polyphénylalanine at any Mg++ concentration. In this paper, we showed that : a) N-acetylphenylalanyl-tRNA did bind to reticulocyte ribosomes in the presence of poly U. This binding was GTP dependent and needed a factor which was extracted by washing ribosomes with salt (NH4CI 1 M); b) the bound N-acetylphenylalanyl-tRNA was partially released by puromycin or by addition of phenylalanyl-tRNA. In this last case, incorporation of N-acetylphenylalanine into polyphenyl- alanine was noticed (ratio N-acetylphenylalanine/phenylalanine = 1/20); c) the ribosomal wash fluid also greatly enhanced phenylalanyl-tRNA binding to salt- treated ribosomes, in the presence of poly U and GTP. The maximum binding was obtained with the same concentrations of Mg++ and K+ as for N-acetylphenylalanyl-tRNA binding. It was not possible to separate on a DEAE-cellulose column the factors that were required for phenyl- alanyl-tRNA and acetylphenylalanyl-tRNA binding. On the other hand, a preparation of the binding enzyme from reticulocyte supernatant enhanced both phenylalanyl-tRNA and acetyl- phenylalanyl-tRNA binding to ribosomes; d) the optimum Mg+÷ concentration for phenylalanine polymerisation was higher with salt- washed ribosomes than with sucrose-washed ribosomes. Adding the wash fluid shifted the Mg++ optimum back to the lower value. N-acetylphenylalanyl-tRNA was not required for this shift as noticed in Escherichia coli. One likely explanation of our results with reticulocytes is the possibility that the ‘binding enzyme’ of Schweet et al., which was present in our ribosome wash fluid, could work with both N-acetyl as well as with phenylalanyl-tRNA. Thus, the inability to incorporate N-acetylphenyl- alanine might be due to a competition between these two compounds for the same enzyme and the same ribosomal site. If this is indeed the case, it is in marked contrast to results obtained with Escherichia coti and could reflect a basic difference in the mechanism of protein chain initiation. [ABSTRACT FROM AUTHOR]

We have previously reported that the catalytic RNA subunit of RNase P of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA[sup Met][sub i]) within the mature tRNA sequence to produce specific fragments. This cleavage was dependent on the occurrence of an altered conformation of the tRNA substrate. We call this further cleavage hyperprocessing. In the present paper, to search for another tRNA that can be hyperprocessed in vitro, we used total mature tRNAs from Drosophila as substrates for the in vitro M1 RNA reaction. We found that some tRNAs can be hyperprocessed by M1 RNA and that two such tRNAs are an alanine tRNA and a histidine tRNA. Using mutant substrates of these tRNAs, we also show that the hyperprocessing by M1 RNA is dependent on the occurrence of altered conformations of these tRNAs. The altered conformations were very similar to that of tRNA[sup Met][sub i]. We show here that M1 RNA can be used as a powerful tool to detect the alternative conformation of tRNAs. The relationship between these hyperprocessing reactions and stability of the tRNA structure will also be discussed. [ABSTRACT FROM AUTHOR]