Figure 1.

(a) Alignment of the amino-acid sequences of cold-shock proteins for which X-ray crystal structures exist. (b) Alignment of the amino-acid sequences of cold-shock proteins from S. typhimurium with the major cold-shock protein from B. subtilis. Identical residues are shown in black, similar residues in grey and non-identical residues in white. Residues involved in DNA binding are marked with asterisks. Ribonucleotide protein motifs 1 and 2 are shown in red and blue, respectively. The abbreviations are as follows: S.T_CSP, cold-shock protein from S. typhimurium; E.C_CspA, cold-shock protein A from E. coli; B.S_CspB, cold-shock protein B from B. subtilis; B.C_Csp, cold-shock protein from B. caldolyticus. (c) Recombinant His₆ StCspE and StCspE resolved on a HiLoad 26/60 Superdex-75 column. The column was calibrated with the molecular-weight standards catalase (232 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa) and vitamin B (1.35 kDa) from Amersham Biosciences. The dashed lines on the trace correspond to the elution maxima of the standards. His₆ StCspE (blue) eluted as a single peak with a retention volume of 13.79 ml, corresponding to a molecular weight of 12.5 kDa. StCspE (after His-tag cleavage; red) eluted as one major and one minor peak with retention volumes of 16.07 and 12.48 ml corresponding to molecular weights of 7.0 and 25 kDa, respectively. (d) SDS–PAGE analysis of 15 µg purified StCspE. (e) Electrophoretic mobility shift assay of StCspE and its complexes with ONc. 15–20 pmol 5′-end γ-³²P-labelled ONc was mixed with increasing amounts of StCspE and incubated for 10 min at 277 K. Amounts of protein: lane 1, 500 pmol; lane 2, 250 pmol; lane 3, 125 pmol; lane 4, 62.5 pmol: lane 5, 31.25 pmol. Lane 6 was a control and contained 5′-end γ-³²P-labelled ONc only. Protein–DNA complexes were separated on a 20% native polyacrylamide gel. (f) StCspE crystal after 24 h of growth. (g) StCspE crystal after 72 h of growth.