Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA

Abstract

Crc is a global modulator involved in Carbon Catabolite Repression (CCR) in Pseudomonas, manipulating the uptake and assimilation of preferred carbon substrates. It is postulated that the free Crc is regulated by a complex and yet unknown process involving small non-coding regulatory RNAs, CrcY and CrcZ. While Crc's interaction with AANAANAA rich target mRNA was proposed initially, but the conclusive proof was never obtained. Recent cryoEM structural studies derived a ternary complex formed by the hexameric RNA chaperon Hfq, Crc, and an mRNA fragment to highlight the assembly of these major elements in P. aeruginosa CCR. Although Crc was shown to kinetically favor and stabilize the association between Hfq with RNA, the interaction between Crc and substrate RNA remains enigmatic. Here, we present the solution structure, dynamics, and function of Crc (∼ 31 kDa, PsCrc) from a psychrotrophic Antarctic bacterium P. syringae Lz4W exclusively using NMR spectrometer operating at 600 MHz 1H frequency. Further, by utilizing several in vitro biophysical and in vivo functional assays to study the interaction between PsCrc and a representative small RNA as well as endogenous CrcY and CrcZ RNA, we demonstrate the significance of residues D100 and K101 in the activity of PsCrc. Our studies suggest that PsCrc has divergently evolved from Apurinic/apyrimidinic (AP) endonuclease by gaining a non-canonical RNA binding region. Our study proposes a dynamic and complex association of PsCrc:CrcY/Z RNA and Hfq:Crc:mRNA to regulate inactive and active CCR. Moreover, the combination of existing solution NMR approaches used in this study paves a path for meaningfully studying high MW proteins with the help of relatively lower field-strength spectrometers.

Keywords

• Crc
• CrcY and CrcZ
• Hfq
• Pseudomonas syringae Lz4W
• backbone dynamics
• and RNA binding protein