Expression, purification, and functional analysis of the TyrR protein of Haemophilus influenzae.
The gene that was inferred to encode the TyrR protein of Haemophilus influenzae Rd was synthesized by polymerase chain reaction and inserted into a T7-based expression vector. Methods were developed to overexpress the TyrR protein of H. influenzae in Escherichia coli and to purify the protein on a large scale. Both in vitro and in vivo functional comparisons of the H. influenzae and E. coli TyrR proteins were carried out. The TyrR protein of H. influenzae was able to bind in vitro to an operator target upstream of the aroF-tyrA gene of E. coli. In the presence of [gamma-S]ATP, the DNA binding ability of the H. influenzae TyrR protein was drastically reduced. Despite the much shorter peptide chain length (318 amino acid residues vs 513), the TyrR protein of H. influenzae was as active in repressing the aroF promoter as the TyrR protein of E. coli. Repression by both proteins was enhanced in the presence of tyrosine; however, the transcriptional activation function associated with the TyrR protein of E. coli could not be detected when the H. influenzae TyrR protein was expressed in E. coli. By computer analysis, at least five operator targets for TyrR were identified within the genomic DNA of H. influenzae. These observations show that the assignment of function to the tyrR gene of H. influenzae was correctly made. Further studies of the H. influenzae TyrR protein in comparison to its E. coli counterpart should provide valuable mechanistic information on transcriptional regulation in this system.
Zhu
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Zhao
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Somerville
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RL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D001483:Base Sequence; D002850:Chromatography, Gel; D029968:Escherichia coli Proteins; D016680:Genome, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D018085:Regulon / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C069256:TyrR protein, E coli; D014443:Tyrosine