Poly(A) sequence of 25 adenylic residues placed immediately before the start codons of the green fluorescent protein (GFP) and firefly luciferase (Luc) mRNAs is shown to provide a high rate of translation of the heterologous messages in eukaryotic cell-free translation systems. Also the poly(A) leader is found to provide the abolition of the inhibition of translation at excess mRNA concentrations. The possibility of the practical use of the constructs with the poly(A) leader for preparative protein production is demonstrated in the wheat germ continuous-exchange cell-free (CECF) translation system.

Ribosomal protein S1 of Thermus thermophilus overexpressed in Escherichia coli cells has been isolated and subjected to studies by analytical sedimentation and differential scanning microcalorimetry techniques. It has been demonstrated that the protein of 60 kDa sediments at s ؊7 cm 2 /s), indicating its compact globular conformation under these conditions. The microcalorimetry study has shown the presence of a cooperative tertiary structure melting at 90°C, but with several (probably three) independent cooperative domains. In the presence of 100 mM NaCl the protein becomes more asymmetric (s 0 20,w ‫؍‬ 3.1 S) but does not lose its cooperativity and thermostability, this suggesting just the weakening of interdomain ionic interactions. The compact globular conformation of protein S1 seems to be most likely within the ribosome.Ribosomal protein S1 is the largest protein of eubacterial ribosomes. It is an RNA-binding protein involved in retention of mRNA during initiation of translation and, maybe, during elongation (1, 2). The Escherichia coli protein S1 was reported to include six copies of a motif of about 70 amino acids (the so-called S1 motifs) separated by spacers of 10 -15 residues (1, 3, 4). Each of these motifs can be arranged into a five-stranded antiparallel ␤-barrel resembling the fold of the bacterial cold shock protein (4). Two N-proximal repeats, however, were found to be rather divergent. At the same time, the N-terminal part of protein S1 was reported to contain the ribosome-binding site of the protein (5), whereas the C-proximal part was claimed to bind mRNA (6). Similar motifs were found also in a number of other RNA-binding proteins and positted to form an ancient nucleic acid-binding fold (4).Physical studies of isolated protein S1 from E. coli showed that the protein in solution manifested a highly extended, noncompact conformation; the data of the sedimentation analysis, small angle x-ray scattering, diffusion coefficient, and intrinsic viscosity measurements, etc., were interpreted in terms of a rod-shaped object of 23 nm in length, which was comparable with the longest dimension of the whole ribosome (1, 7-9).Ribosomal protein S1 has been recently identified in Thermus thermophilus and its gene has been expressed in E. coli (10). As the previous physico-chemical studies of E. coli ribosomal protein S1 demonstrated its highly elongated, asymmetric shape in solution, we decided to reinvestigate this matter with the thermophilic homologue of the protein, bearing in mind that the non-compact state of the isolated E. coli protein could be the consequence of its low physical stability. EXPERIMENTAL PROCEDURESProtein Isolation and Sample Preparation-Recombinant ribosomal protein S1 of T. thermophilus was isolated from the E. coli overproducing strain BL21(DE3)pET21d-tthS1 and purified as described earlier (10). The purity and the quality of the protein were checked by the standard gel electrophoresis method according to Laemmli (11). The protein was stored at 4°C in the precipitated form in (NH 4 ...

Ribosomal protein S1 has been identi¢ed in Thermus thermophilus ribosomes. The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli. A procedure for puri¢cation of the protein has been developed. ß

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