Abstract
Mutations M219K, S266A, and G337S in transcription termination factor Rho have been shown to confer resistance to the antibiotic bicyclomycin (BCM). All three His-tagged mutant Rho proteins exhibited similar Km values for ATP; however, the Vmax values at infinite ATP concentrations were one-fourth to one-third that for the His-tagged wild-type enzyme. BCM inhibition kinetics of poly(C)-dependent ATPase activity for the mutant proteins were non-competitive with respect to ATP (altering catalytic function but not ATP binding) and showed increased Ki values compared with His-tagged wild-type Rho. M219K and G337S exhibited increased ratios of poly(U)/poly(C)-stimulated ATPase activity and lower apparent Km values for ribo(C)10 in the poly(dC).ribo(C)10-dependent ATPase assay compared with His-tagged wild-type Rho. The S266A mutation did not show an increased poly(U)/poly(C) ATPase activity ratio and maintained approximately the same Km for ribo(C)10 in the poly(dC). ribo(C)10-dependent ATPase assay. The kinetic studies indicated that M219K and G337S altered the secondary RNA binding domain in Rho whereas the S266A mutation did not. Transcription termination assays for each mutant showed different patterns of Rho-terminated transcripts. Tyrosine substitution of Ser-266 led to BCM sensitivity intimating that an OH (hydroxyl) moiety at this position is needed for BCM (binding) inhibition. Our results suggest BCM binds to Rho at a site distinct from both the ATP and the primary RNA binding domains but close to the secondary RNA-binding (tracking) site and the ATP hydrolysis pocket.
Highlights
BCM targets Rho transcription termination factor, an essential protein in Escherichia coli [10]
A mutagenesis cartridge was constructed to allow overexpression of mutant Rho protein using the wild-type overexpression system. This was accomplished by replacing the BclI-KpnI DNA fragment from the wild-type plasmid with the BclI-KpnI fragment isolated from M219K, S266A, and G337S
The analysis of BCM inhibition of Rho has led to a model in which BCM interferes with the tracking of Rho at the secondary RNA-binding site [37] and the hydrolysis of ATP but not ATP binding [36]
Summary
BCM was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan), and was further purified by three successive silica gel chromatographies using 20% methanol/chloroform as the eluant [36]. The plasmid pET-RhoW was introduced into BL21(DE3)pLysS, and the expression of Rho protein was induced with addition of 1 mM isopropyl-D-thiogalactopyranoside at a cell concentration of 0.8 A at 600 nm. Construction of Mutant Overexpression Vectors—The rho gene from BCM-resistant E. coli strains BCMr102, BCMr108, and BCMr110 [10] were amplified with primers flanking the BclI and KpnI restriction sites. The resulting amplified DNA was digested with BclI and KpnI and ligated into the BclI-KpnI linearized pET-RhoW plasmid. The plasmids were transformed into BL21(DE3)pLysS, and mutant Rho proteins were overexpressed, as described for the wild-type Rho. Subcloning of rho Gene into Phage M13mp Vector—By using PCR amplification of plasmid DNA containing the wild-type rho gene with two synthetic oligonucleotide primers, a SalI restriction site was introduced 88 bases upstream of the start codon of the rho gene.
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