The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator

Abstract

SummaryThe Crc protein is a global regulator that controls the hierarchical assimilation of carbon sources in Pseudomonads by inhibiting expression of several catabolic pathways. Crc does not bind DNA and its mechanism of action has remained elusive. Among other genes, Crc inhibits expression of alkS, the transcriptional activator of the Pseudomonas putida OCT plasmid alkane degradation pathway. AlkS activates expression of its own gene. In the presence of saturating AlkS levels, translational fusions of alkS to the lacZ reporter gene were responsive to Crc, but transcriptional fusions were not. In translational fusions, the first 33 nt of alkS mRNA, which includes up to position +3 relative to the translation start site, were sufficient to confer an efficient response to Crc. In vitro, purified Crc could bind specifically to an alkS mRNA fragment spanning positions +1 to +43, comprising the translation initiation region. We have previously shown that Crc has little effect on the stability of alkS mRNA. We conclude that Crc modulates AlkS levels by binding to the translation initiation region of alkS mRNA, thereby inhibiting translation. Because AlkS is an unstable protein present in limiting amounts, reducing its levels leads to decreased expression of all genes in the pathway.

The Crc Global Regulator Inhibits the Pseudomonas putida pWW0 Toluene/Xylene Assimilation Pathway by Repressing the Translation of Regulatory and Structural Genes

“…4). Purified Crc can bind in vitro short RNA fragments containing a proper target; the specificity of this binding has been demonstrated by competition and mutagenesis assays (30,31,32). Band-shift assays were therefore used to analyze the ability of purified Crc to bind radioactively labeled run-off RNA fragments corresponding to the 5Ј-end of the mRNAs originating at promoters Pu, Pm, P S1 , and P R1 .…”

“…The latter contains the crc(His 6 ) gene under the control of a T7 promoter (30). Crc protein was purified using a nickel-nitrilotriacetic acid column, as described previously (30).…”

“…Protein Purification-The Crc protein containing a His 6 tag at the C terminus, which is biologically active (30), was overproduced in Escherichia coli BL21(DE3)(pLysS) (39) containing plasmid pCRCH. The latter contains the crc(His 6 ) gene under the control of a T7 promoter (30).…”

The role of FIS protein in the physiological control of the expression of the Escherichia coli meta-hpa operon

“…Bacillus (Saier et al, 1995(Saier et al, , 1996 and Pseudomonas (Cases & de Lorenzo, 1998;Dinamarca et al, 2002;Holtel et al, 1994;Yuste & Rojo, 2001;Petruschka et al, 2001;Sze et al, 1996;Sze & Shingler, 1999), but the molecular mechanisms involved in repression in these micro-organisms are quite different (Cases & de Lorenzo, 2000;Cases et al, 1996Cases et al, , 1999Dinamarca et al, 2002;Moreno et al, 2007).…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…Keeping AlkS levels below those required for maximal induction of the pathway simultaneously inhibits transcription of the alkST and alkBFGHJKL operons. Crc is an RNA-binding protein that interacts with the 5′-end of alkS mRNA, inhibiting its translation [229,230]. Decreasing translation of alkS indirectly reduces alkS transcription because AlkS is less produced and cannot activate the expression of its own gene from promoter PalkS2.…”