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A subset of the spin systems assigned in the ¹H NMR of the trp repressor in the first paper in this series (our penultimate preceding paper in this journal) can be identified as surface or buried residues on the basis of four independent types of measurement: (a) selective spin-lattice relaxation times; (b) the dependence of line widths on temperature and the concentration of manganous ion; (c) fluorescence quenching; and (d) titration behaviour. Criteria are developed for distinguishing surface and buried residues. The significance for the function of DNA biding proteins is discussed. [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]

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]

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the sucking mouse assay respectively. [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]