RESUMEN
The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.
Asunto(s)
Escherichia coli/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Factor 1 Procariótico de Iniciación/metabolismo , Factor 3 Procariótico de Iniciación/metabolismo , Thermus thermophilus/metabolismo , Secuencia de Aminoácidos , Cromatografía , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Operón , Factores de Iniciación de Péptidos/química , Factor 1 Procariótico de Iniciación/química , Factor 3 Procariótico de Iniciación/química , ARN Mensajero/metabolismo , ARN de Transferencia de Metionina/metabolismo , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle. The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry. Thermal transitions of the EF-Tu.GDP and EF-Tu.guanosine-5'-[beta,gamma-imido]triphosphate have a cooperative two-state character. Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu. Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein. Despite the fact that strong interactions between elongation factors Tu and Ts from T. thermophilus at 20 degrees C exist, the thermal transition of neither protein in the complex was significantly affected.