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Chapter 5 : Roles of mRNA Stability, Translational Regulation, and Small RNAs in Stress Response Regulation
Category: Microbial Genetics and Molecular Biology
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The largest class of small RNAs (sRNAs) regulate mRNA stability and translation by pairing with specific target mRNAs. The way in which the mRNA folds can profoundly affect all of its properties. Degradation by RNase E in vitro is known to be stimulated by a 5' monophosphate or 5' OH on the mRNA, even though the initial cleavage event may be far from the 5' end. Initiation of degradation at the 3' end of an mRNA without an internal endonucleolytic cut generally proceeds rapidly only in the absence of secondary structure, such as the stem loop from a factor-independent terminator. Most alterations in mRNA folding by regulatory molecules affect stability or translation, but co-transcriptional changes in folding can also affect mRNA fate via formation or failure to form RNA structures, thereby leading to transcription termination. More recently, it has become apparent that environmental sensing by the 5' end of an mRNA can occur directly, by binding of a small molecule ligand to the 5' UTR. The folding of these mRNA structures, called riboswitches, is modulated by the ligand, leading to transcription termination (OFF switches) or antitermination (ON switches), or translational regulation. Total deletion of CsrA is lethal in Escherichia coli under many growth conditions, apparently because of redirection of the cell to hyperaccumulation of glycogen. Antisense RNAs, known to regulate plasmid stability, have now been found in bacteria and shown to play major regulatory roles.
mRNA stability determinants in E. coli. (A) A variety of features that may improve mRNA stability and (B) cellular processes that lead to mRNA cleavage and degradation. See text for references and details. Light arrows and other symbols show inefficient access of endonucleases and exonucleases to mRNA; darker arrows and symbols indicate better access. No 5′ to 3′ exonucleases are known in E. coli. Degradation is frequently initiated by endonucleases such as RNase E, followed by 3′ to 5′ exonuclease degradation by PNPase, RNase II, and RNase R. mRNA characteristics that protect from RNase E include a 5′-triphosphate; although this can be removed by RppH, secondary structure near the 5′ end may slow or block RppH action. Removal of the 5′ triphosphate increases activity of RNase E in endonucleolytic cleavage of the mRNA. Ribosome loading and translation may also block endonuclease action. 3′ to 5′ exonucleases are either blocked by terminator stem loops or unable to initiate with a short 3′ single-stranded extension; polyA polymerase may add a 3′ extension, allowing these nucleases to enter. Endoribonuclease III makes double-stranded cuts within bulges of stem loops; such cuts are then entry points for the 3′ to 5′ exonucleases.
Regulation at the ends of transcripts: (A) retroregulation and (B) attenuation. (A) In this generalized example, derived from the case of bacteriophage lambda int regulation described in the text, promoter P1 leads to transcripts that end at the factor-independent terminator indicated by the stem loop and run of Us. This is relatively resistant to 3′ to 5′ exonucleases, which cannot access the short single-stranded region at the 3′ end of the transcript; the open reading frame (ORF) mRNA is present and can be translated. Promoter P2 includes an antitermination system that allows the RNA polymerase to read through the terminator, ending somewhere further downstream. This read-through transcript includes a stem loop that is a site for RNase III cleavage; the cleavage product is then sensitive to 3′ to 5′ exonucleases, decreasing mRNA levels and therefore decreasing expression of the ORF. The lightening bolts indicate where environmental or stress information can be sensed, in this case by differential expression of the P1 and P2 promoters. (B) Attenuation and riboswitches: The 5′ UTR of this gene can fold in two alternative structures. On the top line, the light gray sequence pairs with a downstream sequence, forming a terminator, and transcription does not extend into the downstream ORF. This terminator can form because the sequence in the dark heavy sequence is sequestered in the upstream stem loop. On the second line, the upstream sequestering structure does not form, and the dark sequence binds to the grey sequence, blocking formation of a terminator, and allowing transcription to continue into the ORF. Modulators of this folding can include small molecule ligands (as in riboswitches, shown in the top line by the hatched region, also where sensing of the cellular environment takes place), by stalled translation of an upstream leader peptide, or by interactions with uncharged tRNAs for T box regulation (see text).
Hfq trafficking to stimulate sRNA pairing to mRNAs. (A) Hfq binds to single-stranded regions in both sRNAs and mRNA targets. In some cases, binding leads to remodeling of the RNA, increasing the availability of pairing regions. Depicted is the remodeling of the sodB mRNA by Hfq (Geissmann and Touati, 2004 ); the region of sodB that pairs with the sRNA RyhB is shown in grey. Similar remodeling to open up stems has been observed for OxyS (Zhang et al., 2002 ), but remodeling is not detected in other cases of Hfq binding (Brescia et al., 2003 ). (B) It is not yet clear whether two Hfq rings interact, one binding to mRNA (in black) and one to sRNA (in gray), as shown, or if two RNAs bind to a single ring, stabilizing initial pairing. In at least one case, DsrA stimulation of rpoS translation, displacement of Hfq seems to be important to increase pairing, presumably improving activation of translation (Soper and Woodson, 2008 ).