Characterization of proteins of the ribose high-affinity transport system in Escherichia coli K12

Camilla Ann Mauzy, Purdue University

Abstract

The high-affinity transport of ribose in Escherichia coli K12 is accomplished by proteins encoded by the rbs operon. The RbsDAC proteins comprise the putative cytoplasmic membrane transport complex, RbsB is the ribose binding protein, RbsK is ribokinase, and RbsR is the repressor for the operon. A modification of the purification scheme for the ribose binding protein (RBP) was developed using the plasmid pCMB1, which contains the rbsB gene under the control of the lac promoter. Using this construct, RBP can be overproduced to approximately 20% of the total cellular protein. The steps in RBP purification have been reduced to require a chloroform shock step, one ammonium sulfate fractionation step, and two chromatographic steps (gel filtration and anion exchange). The gene for the rbs repressor protein has been sequenced and the translational start site confirmed by protein sequencing. The rbsR gene consists of a 987 base pair coding region producing a 329 residue protein of M$\sb{\rm r}$ 36,369. The start codon for the gene has been determined as "TTG", making rbsR one of only 1.2% of known E. coli genes starting with "TTG". RbsR has been shown to bind to an operator region located between the $-$10 site of the rbs promoter and the start of rbsD. DNAse I footprinting with crude RbsR showed that rbsO was protected from digestion. Methylation interference studies have demonstrated that two sites of RbsR binding exist, one on each face of the DNA. Band shift assays using crude RbsR and rbsO demonstrated that ribose is the inducer molecule. Computational studies using the RbsR sequence have demonstrated that RbsR and other members of this family of repressors are homologous to the ribose binding protein, an indication of the existence of a common protein ancestor. Several experiments were done to demonstrate that RbsA contained ATP binding sites. The rbsA protein was shown to bind specifically to the ATP affinity analogs 8-azido-ATP and 5$\sp\prime$-fluorosulfonylbenzoyl adenosine. Affinity labelled RbsA peptides were isolated and identified as regions located in ATP binding consensus regions. Purified RbsA also demonstrated specific binding affinity for an ATP (C8) agarose column.

Degree

Ph.D.

Advisors

Hermodson, Purdue University.

Subject Area

Biochemistry

COinS