Cloning, sequencing, and expression of dnaK-operon proteins from the thermophilic bacterium Thermus thermophilus

• Published in: Biochimica et biophysica acta 1997-09, Vol.1353 (3), p.253-265
• Main Authors: Osipiuk, Jerzy, Joachimiak, Andrzej
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; ATPase activity; Bacterial Proteins - genetics; Base Sequence; Cloning, Molecular; Conserved Sequence - genetics; dnaK Operon; Escherichia coli - genetics; Escherichia coli Proteins; Gene Expression Regulation, Bacterial - genetics; Genes, Bacterial - genetics; Heat shock protein; Heat-Shock Proteins - genetics; Heat-Shock Response - genetics; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins - chemistry; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - metabolism; Molecular Sequence Data; Molecular Weight; Operon - genetics; Promoter Regions, Genetic - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; RNA, Bacterial - analysis; RNA, Bacterial - biosynthesis; RNA, Messenger - analysis; RNA, Messenger - biosynthesis; Sequence Analysis, DNA; Terminator Regions, Genetic - genetics; Thermophile; Thermus thermophilus - genetics
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(97)00071-7

We propose that the dnaK operon of Thermus thermophilus HB8 is composed of three functionally linked genes: dnaK, grpE, and dnaJ. The dnaK and dnaJ gene products are most closely related to their cyanobacterial homologs. The DnaK protein sequence places T. thermophilus in the plastid Hsp70 subfamily. In contrast, the grpE translated sequence is most similar to GrpE from Clostridium acetobutylicum, a Gram-positive anaerobic bacterium. A single promoter region, with homology to the Escherichia coli consensus promoter sequences recognized by the σ 70 and σ 32 transcription factors, precedes the postulated operon. This promoter is heat-shock inducible. The dnaK mRNA level increased more than 30 times upon 10 min of heat shock (from 70°C to 85°C). A strong transcription terminating sequence was found between the dnaK and grpE genes. The individual genes were cloned into pET expression vectors and the thermophilic proteins were overproduced at high levels in E. coli and purified to homogeneity. The recombinant T. thermophilus DnaK protein was shown to have a weak ATP-hydrolytic activity, with an optimum at 90°C. The ATPase was stimulated by the presence of GrpE and DnaJ. Another open reading frame, coding for ClpB heat-shock protein, was found downstream of the dnaK operon.