Robust Translation of the Nucleoid Protein Fis Requires a Remote Upstream AU Element and Is Enhanced by RNA Secondary Structure

Abstract

Synthesis of the Fis nucleoid protein rapidly increases in response to nutrient upshifts, and Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions. Previous work has shown that control of Fis synthesis occurs at transcription initiation of the dusB-fis operon. We show here that while translation of the dihydrouridine synthase gene dusB is low, unusual mechanisms operate to enable robust translation of fis. At least two RNA sequence elements located within the dusB coding region are responsible for high fis translation. The most important is an AU element centered 35 nucleotides (nt) upstream of the fis AUG, which may function as a binding site for ribosomal protein S1. In addition, a 44-nt segment located upstream of the AU element and predicted to form a stem-loop secondary structure plays a prominent role in enhancing fis translation. On the other hand, mutations close to the AUG, including over a potential Shine-Dalgarno sequence, have little effect on Fis protein levels. The AU element and stem-loop regions are phylogenetically conserved within dusB-fis operons of representative enteric bacteria.

Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria

“…3C). This is reminiscent of the fis (Nafissi et al 2012) and rpsA (Boni et al 2000(Boni et al , 2001Skorski et al 2006) mRNAs that lack true SD-elements; nevertheless, they are translated very efficiently due to the presence of the AU-rich translational enhancers and specific secondary structures within their FIGURE 2. Regulation of the rplY expression at the translation level.…”

“…In E. coli, mRNAs capable of providing a high level of expression in the absence of a canonical SD element are rare: As yet, only rpsA (Boni et al 2001;Skorski et al 2006), fis (Nafissi et al 2012), and rplY (this work) mRNAs can be attributed to this category. The replacement of the rplY GAGAG SD-like sequence by a classic GGAGG-element abolished L25-mediated control and reduced rather than increased the translation yield.…”

“…Many data provide evidence that AU-rich sequences within the mRNA leaders served as targets for the r-protein S1 facilitating ribosome recruitment during initiation (Boni et al 1991(Boni et al , 2001Tzareva et al 1994;Komarova et al 2002Komarova et al , 2005Hook-Barnard et al 2007;Vimberg et al 2007;Nafissi et al 2012). Most likely, the extended AU-rich sequence of the rplY leader functions analogously.…”

Multiple ways to regulate translation initiation in bacteria: Mechanisms, regulatory circuits, dynamics

“…Genes on the same operon may exhibit large differences in their expression and their levels can vary independently (i.e. the dusB-fis operon [181]). More often, they encode for multi-subunit complexes with uneven stoichiometry and their relative levels are the results of very accurate differential translation [182].…”

Synonymous Mutations in rpsT Lead to Ribosomal Assembly Defects That Can Be Compensated by Mutations in fis and rpoA

“…In rpoA, the mutation Leu270Phe in the αCTD domain was found in two independently evolved rpsT mutants carrying the synonymous mutations G48A and A150G, respectively. (Nafissi et al, 2012). The affected nucleotides in the dusB mutants were all located in a predicted stem loop structure, important for ribosome binding and translation of fis.…”