Isolation of Qbeta polymerase complexes containing mutant species of elongation factor Tu.
The RNA genome of coliphage Qbeta is replicated by a complex of four proteins, one of them being the translation elongation factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.
Mathu
Sander G J
SG
Department of Biochemistry, LIC, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
Knudsen
Charlotte R
CR
van Duin
Jan
J
Kraal
Barend
B
D017908:Allolevivirus / Q000201:enzymology*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases; D010444:Peptide Elongation Factor Tu