Cloning, expression, purification and crystallization of the Rho transcription termination factor from Thermotoga maritima.
Rho is an essential ATP-dependent homohexameric helicase that is found in the vast majority of bacterial species. It is responsible for transcription termination at factor-dependent terminators. Rho binds to a specific region of the newly-synthesised mRNA and translocates along the chain until it reaches and disassembles the transcription complex. Basically, two crystallographic structures of Rho hexamer from Escherichia coli have been reported: an open ring with RNA (or ssDNA) bound to the RNA-binding domain, and a closed ring with the RNA bound to both the RNA-binding domain and the ATP-ase domain. The structure of the protein free from RNA is still unknown, but thermophilic bacteria enable an alternative approach to its characterization as their proteins often crystallize more easily than those of their mesophilic homologs. We report here the heterologous expression in E. coli of full-length Rho from the thermophile Thermotoga maritima, a simple protocol for the purification of its hexameric nucleic acid-free form, and the obtainment of 2.4 A-diffracting crystals.
Canals
Albert
A
Institute for Research in Biomedicine and Institut de Biologia Molecular de Barcelona (CSIC), Barcelona Science Park, Baldiri Reixac 10-12, 08028 Barcelona, Spain. albert.canals@irbbarcelona.org
Coll
Miquel
M
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012234:Rho Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020124:Thermotoga maritima / Q000235:genetics / Q000378:metabolism*
D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D012234:Rho Factor