TABLE 2.

Effect of cysteine substitutions in the C-terminal domain of UhpA on DNA binding, transcription activity, and site-specific cleavage

UhpA varianta	Relative protein yieldb	DNA bindingc		Transcription activity			FeBABE cleavagef
		S	W	LacZd	In vitroe
					−CAP	+CAP
Wild type	100	+	+	100	83	100	—
H-WT	100	+	+	300			—
H-A154C	100	+	+	100			—
H-V155C	66	±	−	37
H-G181C	24	+	−	53
H-V182C	33	±	−	38
S183C	<15			63
D185C	43	+	+	51	15	52	W
E187C	100	+	+	70	17	50	S, W
A189C	42	+	+	179	23	92	—
R191C	<10			47

^aUhpA proteins carried the indicated cysteine substitutions. Proteins whose name begins with “H” were synthesized with an N-terminal His tag; all others had the same terminal sequences as the wild-type protein.

^bThe amount of protein, estimated by densitometry of Coomassie blue-stained gels following standard protein purification and resolution by sodium dodecyl sulfate-acrylamide gel electrophoresis, is expressed relative to that of the wild-type protein.

^cBinding of the purified proteins to the uhpT promoter-containing DNA fragment was determined by DNase footprinting assay. Results were expressed as comparable to the wild-type protein for protection of the S and W regions (+), weaker protection than the wild-type (±), or no detectable protection (−).

^dWild-type and cysteine-substitution variants of UhpA protein were expressed from plasmids in cells carrying the uhpT-lacZ fusion. Levels of Glc6P-induced β-galactosidase activities are expressed relative to the wild-type protein.

^euhpT-specific transcription driven by 30 nM RNAP-220 nM UhpA variant, in the presence or absence of 20 nM CAP. The amount of labeled RNA product is expressed as the percent uhpT-specific transcript produced by the wild-type UhpA protein.

^fDNA cleavage of the S or W regions following incubation of DNA with FeBABE-modified UhpA proteins in the presence of hydrogen peroxide and ascorbate. Enhanced cleavage in the S or W regions is indicated. —, no enhanced cleavage.