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.
Highlights
Hfq-binding small RNAs, major regulatory RNAs in bacteria, are induced under specific physiological and/or stress conditions and regulate, along with an RNA chaperone Hfq, the expression of target genes at the post-transcriptional level (Waters and Storz 2009; Gottesman and Storz 2010; Vogel and Luisi 2011; Wagner and Romby 2015)
We demonstrated previously by using a “double terminator system” that RNA polymerase frequently reads through the Rho-independent terminator of sgrS under normal growth conditions (Morita et al 2015)
We have previously found that the poly(U) tail must be seven residues or longer to achieve a stable binding to Hfq that is required for the regulatory function of sRNAs (Otaka et al 2011)
Summary
Hfq-binding small RNAs (sRNAs), major regulatory RNAs in bacteria, are induced under specific physiological and/or stress conditions and regulate, along with an RNA chaperone Hfq, the expression of target genes at the post-transcriptional level (Waters and Storz 2009; Gottesman and Storz 2010; Vogel and Luisi 2011; Wagner and Romby 2015). The primary role of Hfq is to accelerate base-pairing between sRNAs and target mRNAs to regulate their translation. The second role of Hfq is to recruit RNase E near target mRNAs leading to rapid degradation of sRNA–mRNA hybrids (Massé et al 2003; Morita et al 2005). Hfq plays a role in stabilization of sRNAs by protecting them from the attack of ribonucleases (Massé et al 2003). The promoter of the individual sRNA gene is under the control of at least one transcription factor that is modulated by the cognate stress. SgrS and RyhB of Escherichia coli are among well-characterized sRNAs. In response to the glucose-phosphate stress, such as accumulation of glucose-6-phosphate, a transcription factor SgrR is activated to stimulate the transcription of sgrS encoding
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