RRC ID 33366
Author Richardson LV, Richardson JP.
Title A vector for controlled, high-yield production of specifically mutated proteins in Escherichia coli: test of a putative cytidine-binding domain in Rho factor and its Thr16----Ala mutant.
Journal Gene
Abstract A derivative of the plasmid vector, pET-3a [Rosenberg et al., Gene 56 (1987) 125-135], is described that contains the origin of replication from bacteriophage f1. This plasmid is well-suited for oligodeoxyribonucleotide mutagenesis and controlled production of mutant proteins from a single vector. Its utility is demonstrated by the preparation of a mutational alteration of Thr16----Ala (T16A) of the Escherichia coli transcription termination factor, Rho. The altered protein (T16A Rho) binds oligo(C)7 with the same affinity as wild-type (wt) Rho, thus indicating that Thr16 is not critical for binding cytidine residues in RNA, in spite of its being part of a sequence that is similar to a sequence in the CTP-binding domain of aspartate transcarbamoylase. However, T16A Rho was less efficient in terminating transcription than was wt Rho and had a lowered kcat for ATP hydrolysis with cro RNA as co-factor. Thus, the change affects the coupling of ATP hydrolysis by Rho to actions on RNA that cause termination.
Volume 118(1)
Pages 103-7
Published 1992-9-1
PII 0378-1119(92)90255-N
PMID 1511872
MeSH Base Sequence Binding Sites Cytidine / metabolism Escherichia coli / genetics* Genetic Vectors / genetics* Molecular Sequence Data Mutagenesis, Site-Directed / genetics* Mutation Oligoribonucleotides / metabolism Peptide Chain Termination, Translational / genetics Recombinant Proteins / biosynthesis Recombinant Proteins / genetics Rho Factor / biosynthesis Rho Factor / genetics* Transcription, Genetic / genetics*
IF 2.498
Times Cited 14
Prokaryotes E. coli pET3af1