Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain

Abstract

The Escherichia coli L-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of L-rhamnose. Here, we examined the ability of the DNAbinding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of L-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ⌬rhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.The RhaS protein functions to activate transcription of two of the operons in the Escherichia coli L-rhamnose regulon in response to the availability of L-rhamnose (11,40). The two operons rhaBAD and rhaT encode the L-rhamnose catabolic enzymes (L-rhamnulokinase, L-rhamnose isomerase, and Lrhamnulose-1-phosphate aldolase) (2, 25) and an L-rhamnoseproton symporter that is responsible for transporting L-rhamnose into the cell (35), respectively. RhaS is encoded in an operon that also encodes a second L-rhamnose-responsive transcription activator, RhaR (37). RhaR activates transcription of the operon that encodes the two activator proteins, rhaSR (37, 38). All three operons in the L-rhamnose regulon also require a second activator protein, cyclic AMP (cAMP) receptor protein (CRP), for full transcription activation (11,16,40).

The AraC/XylS Family Activator RhaS Negatively Autoregulates rhaSR Expression by Preventing Cyclic AMP Receptor Protein Activation

“…Given that full-length RhaS protein severely aggregates when overexpressed, we used the purified C-terminal DNA binding domain of RhaS [His6-RhaS(163-278), previously called His6-RhaS-CTD (34)] for these assays. His6-RhaS(163-278) was previously found capable of in vitro DNA binding and transcription activation (34). Using electrophoretic mobility shift assays, we found that His6-RhaS(163-278) was able to bind in vitro to DNA fragments carrying each of the RhaR half sites at rhaSR, although 3-to 10-fold less tightly than to the RhaS half sites at rhaBAD (Fig.…”

“…To further test this hypothesis, we assayed a fusion with an upstream endpoint at Ϫ312 and carrying point mutations at three consensus positions in the CRP binding site ( Fig. 1) that we previously found greatly reduced CRP coactivation of rhaSR (34). We found that the CRP binding site point mutations reduced RhaS repression of the ⌬312 fusion by more than sixfold to a level similar to that of the ⌬114 fusion (Table 1, ⌬312 CRP Ϫ ).…”

“…However, the promoter-proximal RhaR half site at rhaSR is identically positioned relative to the promoter as the similar RhaS half site at rhaBAD, suggesting that RhaS might activate rather than repress transcription from this site. To test whether RhaS was able to activate rhaSR expression, we assayed expression from single-copy rhaS-lacZ fusions in a strain lacking RhaR (⌬rhaSR strain background), with RhaS expressed from the vector pSU18 (2) (plasmid pSE273 [34] (34). In the ⌬rhaS rhaR ϩ strain, the vector-only samples represent the level of activation by RhaR, while the addition of the RhaS-encoding plasmid shows the RhaS-mediated reduction of the RhaR activation (Fig.…”

“…The 49-bp DNA fragments each contained one 17-bp RhaS or RhaR binding half site (rhaI 1 , rhaI 2 , rhaI 3 , or rhaI 4 ) flanked by the same 16-bp sequences for each half site. The flanking sequences were the same as the previously published half-site fusions with lacZ (34). The mobility shift assay buffer did not contain Nonidet P-40 or cyclic AMP and contained 0.5 mM Tris[2-carboxyethyl]-phosphine (TCEP) instead of 1 mM dithiothreitol.…”

“…The mobility shift assay buffer did not contain Nonidet P-40 or cyclic AMP and contained 0.5 mM Tris[2-carboxyethyl]-phosphine (TCEP) instead of 1 mM dithiothreitol. His6-RhaS(163-278) was purified as previously described (34). After electrophoresis, the gels were dried, exposed to a phosphor screen, and analyzed using the Cyclone storage phosphor system (Packard).…”