Previous work by Schulman and Pelka (1975) J. Biol. Chem. 250, 542-547, indicated that the absence of a pairing between the bases 1 and 72 in initiator tRNA(fMet) explained the relatively small activity of peptidyl-tRNA hydrolase towards N-acetyl-methionyl-tRNA(fMet). In the present study, the structural requirements for the sensitivity of an N-acetyl-aminoacyl-tRNA to Escherichia coli peptidyl-tRNA hydrolase activity have been further investigated. Ten derivatives of tRNA(fMet) with various combinations of bases at positions 1 and 72 in the acceptor stem have been produced, aminoacylated and chemically acetylated. The release of the aminoacyl moiety from these tRNA derivatives was assayed in the presence of peptidyl-tRNA hydrolase purified from an overproducing strain. tRNA(fMet) derivatives with either C1A72, C1C72, U1G72, U1C72 or A1C72 behaved as poor substrates of the enzyme, as compared to those with C1G72, U1A72, G1C72, A1U72 or G1U72. With the exception of U1G72, it could be therefore concluded that the relative resistance of tRNA(fMet) to peptidyl-tRNA hydrolase did not depend on a particular combination of nucleotides at positions 1 and 72, but rather reflected the absence of a base pairing at these positions. In a second series of experiments, the unpairing of the 1 and 72 bases, created with C-A or A-C bases, instead of G-C in methionyl-tRNA(mMet) or in valyl-tRNA(Val1), was shown to markedly decrease the rate of hydrolysis catalysed by peptidyl-tRNA hydrolase. Altogether, the data indicate that the stability of the 1-72 pair governs the degree of sensitivity of a peptidyl-tRNA to peptidyl-tRNA hydrolase.

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A second thioredoxin, Chl, distinct from the one recently reported [Decottignies, P., Schmitter, J. M., Jacquot, J. P., Dutka, S., Picaud, A. & Gadal, P. (1990) Arch. Biochem. Biophys. 280, 112-1211 has been purified from the green alga, Chlamydomonas reinhardtii, and its functional and structural properties investigated. Its activity in various enzymatic assays has been compared with the activities of different plant thioredoxins (Ch2 from C. reinhardtii and spinach m andfl. Chl cannot serve as a substrate for Escherichia coli thioredoxin reductase, but can be reduced by spinach ferredoxin-thioredoxin reductase. It is less efficient than its spinach counterpart in the activation of corn leaf NADP-dependent malate dehydrogenase by light or dithiothreitol, and it only activates spinach fructose-l,6-bisphosphatase at very high concentrations. The complete primary structure of C. reinhardtii thioredoxin Chl was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin and Staphylococcus aureus V8 protease digestions. When needed, peptide masses were verified by plasma desorption mass spectrometry. Chl consists of a polypeptide of 11 1 amino acids (1 1 634 Da) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. Compared to thioredoxins from other sources, the algal thioredoxin Chl displays few sequence similarities with all the thioredoxins sequenced so far. Preliminary evidence indicates that Chl may be an h-type thioredoxin.In all photosynthetic organisms investigated to date, light plays an important role in the regulation of the activity of several chloroplast enzymes through the ferredoxin-thioredoxin system [l]. In this system, the disulfide bridge of thioredoxin is reduced by a specific ferredoxin-thioredoxin reductase in the presence of photoreduced ferredoxin [2]. The last step is the reduction of a disulfide bond on a target enzyme. Two different thioredoxins have been described in spinach leaf chloroplasts: thioredoxin m, preferentially activating NADP-dependent malate dehydrogenase (NADP-MDH) and thioredoxinf, specifically activating fructose-l,6-bisphosphatase [Fru(l ,6) In addition to being present in plants, thioredoxins have been found in several other organisms [7]. The active-site Correspondence to P.