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Translational Coupling to an Upstream Gene Promotes Folding of the Mycobacterial Plasmid pAL5000 Replication Protein RepB and Thereby Its Origin Binding Activity

Department of Microbiology, Bose Institute, Kolkata 700054, India

Received 10 July 2003/ Accepted 20 October 2003

In the mycobacterial plasmid pAL5000 replication region, the replication genes repA and repB are organized in an operon. Earlier, a RepB-dependent origin binding activity was detected in Escherichia coli cells expressing the repA-repB operon. This activity was maximal when expression of the two genes was coupled (A. Basu, M. Chawla-Sarkar, S. Chakrabarti, and S. K. Das Gupta, J. Bacteriol. 184:2204-2214, 2002). In this study we have shown that translational coupling makes a significant difference in the structure and function of RepB. When repB expression was coupled to repA, the polypeptide folded into an active structure (referred to as RepB*), which possessed higher helical content than RepB expressed independently. RepB* could also be distinguished from the less active RepB on the basis of sensitivity to OmpT, an outer membrane protease of E. coli: RepB* was sensitive to the protease, whereas RepB was resistant. Similar conformational differences between RepB* and RepB could be observed when repA was replaced with an unrelated gene, malE (encoding maltose binding protein). These results show that translational coupling of repB to an upstream gene is necessary for better folding and origin binding activity. It is speculated that in coupled systems where translation machinery is passed on from the upstream to the downstream open reading frame, cotranslational folding of the polypeptide expressed from the downstream open reading frame is enhanced due to increased folding competence of translationally primed ribosomes.

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