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.
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.
Richardson
L V
LV
Department of Chemistry, Indiana University, Bloomington 47405.
Richardson
J P
JP
D001483:Base Sequence; D001665:Binding Sites; D003562:Cytidine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation; D009843:Oligoribonucleotides / Q000378:metabolism; D010443:Peptide Chain Termination, Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012234:Rho Factor / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
D009843:Oligoribonucleotides; D011994:Recombinant Proteins; D012234:Rho Factor; D003562:Cytidine