Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs

Abstract

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3 ′ ′ ′ ′ ′ end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs.

Trans-Acting Small RNAs and Their Effects on Gene Expression in Escherichia coli and Salmonella enterica

“…However, further work led to evidence for codegradation of sRNAs with mRNAs (59,91). Regulation, both positive and negative, could also include other forms of processing such as RNA modification or degradation of the U tail; the latter is expected to greatly reduce Hfq binding (92).…”

“…There are also examples of sRNAs such as SroC, where the sRNA is derived from the intergenic region of an operon and must be generated by transcription termination that only occurs part of the time, such that the downstream genes, gltJKL, are also transcribed (97). Since the shorter 3′ U tails or readthrough past the U residues reduce or eliminate Hfq binding, changes in the sequence of the Rhoindependent terminator or growth conditions that change transcription termination can impact sRNA activity (92).…”

The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs

“…The majority of sRNAs, called canonical or Class I sRNAs, recognize the proximal and rim surfaces of Hfq, while their target mRNAs bind to the distal face (Mikulecky et al 2004;Sauer et al 2012;Schu et al 2015). The proximal face of Hfq binds the 3 ′ -terminal oligouridine tails of these sRNAs (Otaka et al 2011;Sauer and Weichenrieder 2011;Ishikawa et al 2012;Morita et al 2017), while the positively charged patches on the rim of Hfq contact the adenosine and uridine-rich sequences present in the central or 5 ′ -terminal regions of sRNA molecules (Sauer et al 2012;Dimastrogiovanni et al 2014). The distal face of Hfq recognizes adenosine-rich motifs, including repeated ARN sequences, which are present in mRNAs regulated by these sRNAs (de Haseth and Uhlenbeck 1980;Mikulecky et al 2004;Soper and Woodson 2008;Link et al 2009;Soper et al 2011;Peng et al 2014a,b;Wroblewska and Olejniczak 2016;Chen and Gottesman 2017;Andrade et al 2018).…”

The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA

“…The PrrF sRNAs were previously shown to interact with the P. aeruginosa Hfq protein (34), indicating a role for Hfq in PrrF regulatory function. Studies of E. coli and Salmonella sRNAs showed that the 3=-terminal U's associated with the Rho-independent transcription terminator bind the proximal pore of Hfq (45,46). An internal AU-rich motif additionally interacts with the arginine patch on the rim of Hfq (47,48), which is necessary for efficient annealing with its mRNA targets (36).…”