ABSTRACT
CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of
Escherichia coli
. Here we identified a novel mechanism in which CsrA represses
iraD
translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and
lacZ
fusions demonstrated that CsrA represses
iraD
expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the
iraD
leader transcript, all of which are far upstream of the
iraD
ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the
iraD
Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the
iraD
leader region. Two of these ORFs do not affect
iraD
expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the
iraD
start codon, a sequence arrangement indicative of translational coupling.
In vivo
expression and
in vitro
translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of
iraD
translation occurs entirely via translational coupling with this ORF, leading to accelerated
iraD
mRNA decay.
IMPORTANCE
CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of
iraD
, which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the
iraD
leader transcript but does not directly occlude ribosome binding to the
iraD
SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of
iraD
, causing repression of
iraD
translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate
csrA
transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.
A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3
