In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and cpsB::lacZ assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.

The Escherichia coli ClpYQ (HslUV) complex is an ATP-dependent protease, and the clpQ+Y+ (hslV+U+) operon encodes two heat shock proteins, ClpQ and ClpY, respectively. The transcriptional (op) or translational (pr) clpQ+::lacZ fusion gene was constructed, with the clpQ+Y+ promoter fused to a lacZ reporter gene. The clpQ+::lacZ (op or pr) fusion gene was each crossed into lambda phage. The λclpQ+::lacZ+ (op), a transcriptional fusion gene, was used to form lysogens in the wild-type, rpoH or/and rpoS mutants. Upon shifting the temperature up from 30 °C to 42 °C, the wild-type λclpQ+::lacZ+ (op) demonstrates an increased β-galactosidase (βGal) activity. However, the βGal activity of clpQ+::lacZ+ (op) was decreased in the rpoH and rpoH rpoS mutants but not in the rpoS mutant. The levels of clpQ+::lacZ+ mRNA transcripts correlated well to their βGal activity. Similarly, the expression of the clpQ+::lacZ+ gene fusion was nearly identical to the clpQ+Y+ transcript under the in vivo condition. The clpQm1::lacZ+, containing a point mutation in the -10 promoter region for RpoH binding, showed decreased βGal activity, independent of activation by RpoH. We conclude that RpoH itself regulates clpQ+Y+ gene expression. In addition, the clpQ+Y+ message carries a conserved 71 bp at the 5’ untranslated region (5’UTR) that is predicted to form the stem-loop structure by analysis of its RNA secondary structure. The clpQm2Δ40::lacZ+, with a 40 bp deletion in the 5’UTR, showed a decreased βGal activity. In addition, from our results, it is suggested that this stem-loop structure is necessary for the stability of the clpQ+Y+ message.