Operator recognition by the ROK transcription factor family members, NagC and Mlc

Abstract

NagC and Mlc, paralogous members of the ROK family of proteins with almost identical helix-turn-helix DNA binding motifs, specifically regulate genes for transport and utilization of N-acetylglucosamine and glucose. We previously showed that two amino acids in a linker region outside the canonical helix-turn-helix motif are responsible for Mlc site specificity. In this work we identify four amino acids in the linker, which are required for recognition of NagC targets. These amino acids allow Mlc and NagC to distinguish between a C/G and an A/T bp at positions ±11 of the operators. One linker position, glycine in NagC and arginine in Mlc, corresponds to the major specificity determinant for the two proteins. In certain contexts it is possible to switch repression from Mlc-style to NagC-style, by interchanging this glycine and arginine. Secondary determinants are supplied by other linker positions or the helix-turn-helix motif. A wide genomic survey of unique ROK proteins shows that glycine- and arginine-rich sequences are present in the linkers of nearly all ROK family repressors. Conserved short sequence motifs, within the branches of the ROK evolutionary tree, suggest that these sequences could also be involved in operator recognition in other ROK family members.

BadR (BB0693) controls growth phase‐dependent induction of rpoS and bosR in Borrelia burgdorferi via recognizing TAAAATAT motifs

“…Typically, E. coli NagC and Mlc proteins bind to DNA through a ‘TT‐9 bp‐AA’ motif; in the central 9‐bp region, there is a very strong preference for ‘CGCGNCGCG’ (Plumbridge, ; Brechemier‐Baey et al ., 2012; 2015). However, these characteristics are not conserved in PrpoS or UPbosR.…”

“…BadR contains only two residues (G153 and H243) conserved in the NagC or XylR SDGs, supporting our conclusion that BadR probably does not bind specifically to GlcNAc‐6P or Xylulose‐5P (our EMSA data revealed that the binding of rBadR to PrpoS or UPbosR was not affected by phosphorylated sugars such as GlcNAc‐6P, Glucose‐6P and xylulose‐5P). Third, PrpoS or UPbosR does not contain a NagC or XylR binding consensus sequence (Jacob et al ., ; Stulke and Hillen, ; Brechemier‐Baey et al ., 2012; 2015). Finally, expression of GlcNAc metabolic genes such as nagA and nagB was not affected in a badR mutant (Miller et al ., ), implying that BadR does not act as bacterial NagC protein.…”

“…Typically, the interaction between bacterial ROK repressors and DNA targets is interrupted by phosphorylated sugars. For example, E. coli NagC functions as a repressor to control expression of genes involved in the uptake and utilization of Nacetylglucosamine (GlcNAc) by responding to Nacetylglucosamine-6-phosphate (GlcNAc-6P); the binding of NagC to DNA is specifically inhibited by GlcNAc-6P (Brechemier-Baey et al, 2015). B. subtilis XylR negatively controls the xylose utilization pathway with xylose acting as an inducer or effector (Gartner et al, 1988;Sizemore et al, 1992;Dahl et al, 1994;Scheler and Hillen, 1994).…”

Impact of Combined Acidic and Hyperosmotic Shock Conditions on the Proteome ofListeria monocytogenesATCC 19115 in a Time-Course Study

“…NagC (lmo0032) was induced by up to 1.15, 0.93, and 0.95 log 2 -fold at the analyzed time points (Table 1 and Figure 3). is protein is most likely a ROK family repressor for the adjacent carbohydrate uptake and metabolism system (lmo0033-0035) though the compounds metabolised remain largely uncharacterised [49,50].…”

Characterization of DNA Binding Sites of RokB, a ROK-Family Regulator from Streptomyces coelicolor Reveals the RokB Regulon

“…glutamicum ATCC 13032 has a similar organisation [ 12 ], as well as the binding sites of Mlc and NagC from E . coli [ 35 ], the binding site of RafR from B . breve UCC2003 [ 15 ], or all described binding sites from the phylum Thermotogae [ 18 ].…”