Part:BBa_K1602049
RRkey
This part is half of a two-part riboregulator-system for E.coli for posttransciptional regulation of gene expression. Upon transcription the produced trans-activating RNA-sequence (taRNA) forms a RNA-RNA-complex with corresponding cis-repressing sequences (crRNA). This leads to a helix shift and the release of the formerly masked ribosome binding site (RBS) enabling the expression of the regulated gene of interest (GOI). The corresponding crRNAs are RRlocked, RRlocked_site and RRGFP.
The sequence of the taRNA is based on an existing riboregulator sequence pair published by Isaacs et al[1]. The original sequence contained an EcoRI restriction site which was removed by basepair-exchange. We used the Riboswitch Designer to find out which basepair-exchange is best suited to remove the unwanted EcoRI restriction site while not affecting the folding- and interaction-capabilities of the sequence.
Functional Parameters
References
1. Isaacs, F.J., et al., Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol, 2004. 22(7): p. 841-7.
Contribution: XHD-Wuhan-Pro-China 2021
literature1
RNA degradation counterbalances transcription, and therefore plays an important regulatory role in adjusting the steady-state level of a given mRNA. Unlike stable ribosomal and transfer RNAs, many mRNAs are labile and their decay is frequently initiated shortly after or, in peculiar cases, even before their transcription is completed (Cannistraro & Kennell, 1985). In Escherichia coli, and presumably in many other Gram-negative bacteria, the rate of mRNA decay is dependent on initial cleavage(s) mainly performed by endoribonuclease RNase E (Melefors, 1993; Kushner, 2002), sometimes by RNase III (Conrad & Rauhut, 2002) or other endoribonucleases (Arraiano, 1993; Alifano, 1994; Arraiano, 1997; Umitsuki, 2001; Li & Altman, 2003). RNase E cleaves RNAs with a 5′ monophosphate group preferentially over those endowed with a 5′ triphosphate (Lin-Chao & Cohen, 1991; Mackie, 1998, 2000). Recent structural data (Callaghan, 2005) confirm the previous finding (Jiang, 2000) that the structural module discriminating mono- and triphosphate groups is located within the catalytic domain of RNase E. Because primary transcripts possess triphosphate groups, whereas the downstream products of RNase E cleavage are monophosphorylated (Misra & Apirion, 1979), the ‘5′-end dependency’ of RNase E appears to pave the way for efficient cleavage at any RNase E recognition site downstream of the initial cleavage (Mackie, 1998, 2000). Although some Gram-positive bacteria lack RNase E sequence homologues (Condon & Putzer, 2002), they apparently have functional counterparts. Putzer and coworkers have recently identified the Bacillus subtilis RNase J1/RNase J2 endoribonucleases (Even, 2005) that biochemically resemble E. coli RNase E.
In E. coli, RNase E provides a scaffold for a multienzyme complex termed the ‘degradosome’ that, in addition to RNase E, contains polynucleotide phosphorylase (PNPase), a 3′→5′ exoribonuclease, the RhlB RNA helicase, the glycolytic enzyme enolase, and a number of minor components (Carpousis, 2002). Several lines of evidence suggest the physical and functional cooperation of degradosomal proteins in the degradation and processing of E. coli RNA in vivo (Xu & Cohen, 1995; Lopez, 1999; Liou, 2001; Leroy, 2002; Bernstein, 2004; Khemici & Carpousis, 2004;,Khemici, 2005). It is commonly accepted that the ‘5′-end dependency’ of RNase E together with the concerted action of the key ribonucleases and auxiliary enzymes such as RhlB and poly(A) polymerase I lead to efficient decay of E. coli transcripts after their initial cleavage by RNase E. As many details of the mRNA decay machinery are not covered here, the reader is referred to previous reviews on this topic (Carpousis, 1999; Coburn & Mackie, 1999; Régnier & Arraiano, 2000; Steege, 2000; Dreyfus & Regnier, 2002; Kushner, 2002).
The relatively short 3′ untranslated regions of bacterial mRNA(s) frequently consist of stable stem–loop structures that can serve as barriers against 3′→5′ exonucleases. In contrast, 5′ untranslated region(s) [UTR(s)] can bear regulatory elements that are involved in down- or up-regulation of translation with corresponding effects on mRNA stability. Here, we focus primarily on regulatory circuits operating within 5′ UTRs and specifically discuss the role of ribosome binding signals, translational repressors, low molecular weight effectors, noncoding RNAs (ncRNAs) and temperature as parameters affecting mRNA stability (Fig. 1a). For additional mechanisms linking translation elongation/termination and mRNA stability including ribosome pausing that leads to mRNA cleavage by interferases, a family of ribonucleases encoded by several toxin–antitoxin systems, the reader is referred to recent reviews (Deana & Belasco, 2005; Condon, 2006).
Reference
Kaberdin VR, Bläsi U. Translation initiation and the fate of bacterial mRNAs. FEMS Microbiol Rev. 2006 Nov;30(6):967-79. doi: 10.1111/j.1574-6976.2006.00043.x. Epub 2006 Sep 21. PMID: 16989654.
Literature2
5′ untranslated regions of many bacterial mRNAs serve as elements controlling the fate of transcripts. (a) Factors influencing the fate of bacterial mRNAs. Indicated are regulatory factors (RNA-binding proteins, noncoding regulatory RNAs (ncRNAs), low molecular weight (LMW) effectors, endoribonucleases (RNase E and RNase III) and 30S subunits). (b) Endonucleolytic cleavage(s) within the 5′ untranslated region of a bacterial mRNA (shown on the left) or factor binding to a region overlapping with the ribosome binding site (shown on the right) which inhibits translation, and subsequently leads to mRNA decay mediated by RNase E and other endo- and exoribonucleases.
Ribosome binding and mRNA stability
The common translation initiation pathway in bacteria, which leads to ternary complex formation between mRNA, fMet-tRNAfMet and the 30S ribosomal subunit, is kinetically controlled by three translation initiation factors, IF1, IF2 and IF3, respectively. The molecular interactions involved in this process encompass base-pairing of the Shine and Dalgarno (SD) sequence on mRNA with the anti-SD sequence residing at the 3′ end of 16S rRNA gene and the interaction of the start codon with the anticodon of fMet-tRNAfMet, respectively (Gualerzi & Pon, 1990; Gualerzi, 2001). In general, inefficient ribosome binding to the 5′ ends of canonical mRNAs has been shown to decrease their stability (Wagner, 1994; Iost & Dreyfus, 1995; Arnold, 1998). Naturally occurring examples of mRNAs with a poor ribosome binding efficiency include leaderless mRNAs, which contain a 5′ terminal start codon and lack canonical ribosome recruitment signals (Moll, 2002). One study (Baumeister, 1991) has demonstrated that the low translational efficiency of the leaderless Tn1721 tetR mRNA is associated with a short half-life.
As mentioned above, the decay of canonical mRNAs is frequently initiated by RNase E cleavages in the 5′ UTR of mRNA (Melefors & von Gabain, 1988; Gross, 1991). RNase E cleaves A/U-rich regions (Lin-Chao, 1994; McDowall, 1994) that preferentially have a G positioned two nucleotides upstream from the scissile bond (Kaberdin, 2003; Redko, 2003). Such A/U-rich sequences are frequently found in the vicinity of the ribosome binding site (RBS) of E. coli mRNAs, and random cloning approaches have revealed that these sequences stimulate translation when placed up- (Dreyfus, 1988) or downstream (Qing, 2003) of a start codon. One rationale for these findings is that A/U-rich regions keep the translation initiation region unstructured and, thereby, facilitate ribosome binding. While interacting with the translation initiation region, the 30S ribosome subunit confers protection to an c. 50-nt region bracketing the start codon (Hüttenhofer & Noller, 1994). Hence, a ribosome bound at the RBS would be anticipated to protect A/U-rich regions flanking the start codon from RNase E cleavage. In support of this idea, in vitro experiments with E. coli ompA mRNA have shown that 30S ribosomal subunits bound to the RBS can protect from RNase E cleavage in the 5′ UTR (Vytvytska, 2000). Moreover, mutations that extend the complementarity between Shine–Dalgarno (SD) and anti-SD sequences increased translation and mRNA stability in vivo (Wagner, 1994; Iost & Dreyfus, 1995; Arnold, 1998). Thus, it is tempting to speculate that a rate-limiting step in mRNA decay depends on the binding affinity of either the 30S ribosomal subunit or RNase E for a given mRNA.
A candidate for a ribosomal component that protects from RNase E cleavage is protein S1. This ribosomal protein is known to contribute to the 30S/mRNA interaction in E. coli (Boni, 1991; Komarova, 2005). Cryo-electron microscopic studies (Sengupta, 2001) revealed that S1 interacts with 11 nucleotides located immediately upstream of the SD sequence. S1 binding to this region is considered of importance for 30S ribosomal recognition of the 5′ region of E. coli mRNAs and/or for stabilization of the ternary complex (Boni, 1991). Experiments performed in our laboratory showed that S1 binding sites and RNase E cleavage sites can coincide on different mRNA substrates, suggesting that ribosome binding to the translation initiation region might involve protection of RNase E cleavage sites by S1. Proximal RNase E cleavage upstream of the RBS (i.e. within a S1 binding site) is not only expected to diminish or eventually prevent further ribosome loading, but would also generate a 5′ monophosphate group at the 5′ end of the processed mRNA, which in turn is expected to stimulate RNase E cleavages within the ribosome-free, downstream region (Fig. 1b, left). The location of coincident S1 binding and RNase E cleavage sites upstream of the SD sequence could provide a means to eliminate selectively mRNAs that are in a translationally ‘inactive conformation’, e.g. where the SD sequence and/or the start codon are sequestered by secondary structure. This would diminish ribosome loading, whereas RNase E cleavage could be unaffected. Moreover, when the concentration of free 30S ribosomal subunits becomes limiting, endonucleolytic cleavages at the 5′ end could provide a mechanism to redirect ribosomes to transcripts, the 5′ UTRs of which are intrinsically more resistant to the nucleolytic activity of RNase E.
Recent studies revealed that ribosome binding to the translation initiation region per se is not sufficient to protect the entire body of E. coli mRNAs from degradation (Arnold, 1998; Joyce & Dreyfus, 1998). Joyce & Dreyfus (1998) demonstrated that, in the absence of translation, lacZ mRNA was not stabilized by the presence of a long SD sequence at its 5′ end. Likewise, it has been recently found that ongoing translation rather than ribosome binding per se is required to protect an ompA-bla fusion mRNA from degradation (Arnold, 1998), and the stabilizing effect of ribosome traffic apparently depends on the length of translated segments of this transcript (Nilsson, 1987).
A recent study by the Boni group (Komarova, 2005) provided further support for these findings. The introduction of A/U-rich sequences upstream of the SD increased translation and the stability of lacZ mRNA, which can be reconciled with the idea that the creation of S1-binding sites accelerates the forward kinetics of ribosome binding and consequently coverage of the body of the mRNA by elongating 70S particles (Komarova, 2005). As further discussed below, mRNA decay in E. coli can thus be considered a consequence of translational inhibition, i.e. in the absence of ribosome traffic along the mRNA it becomes vulnerable to endonucleolytic attack (Fig. 1b, on the left).
In contrast to E. coli mRNAs, several mRNAs of Gram-positive bacteria such as B. subtilis phage SP82 RNA (Hue, 1995) and B. thuringiensis cryIIIA toxin RNA (Agaisse & Lereclus, 1996) have been proposed to be stabilized by either binding or stalling of ribosomes near the 5′ end. In both cases canonical SD-sequences function as stabilizers of the downstream segments, and ribosome binding might protect the 5′ ends of the transcripts from degradation by nucleases. In agreement, the phage SP82 stabilizer conferred increased stability to several heterologous mRNAs when inserted at their 5′ end (Hue, 1995). Further experiments demonstrated the importance of translation initiation complex formation at the 5′end of mRNAs, rather than the transit of ribosomes along the mRNA in B. subtilis (Sharp & Bechhofer, 2003), leading the authors to suggest that ribosome binding at the 5′ end of the mRNA interferes with a 5′-end dependent activity, possibly a 5′-binding endonuclease such as RNase J1 and/or RNase J2 (Even, 2005).
Control of translation in Gram-positive bacteria can also involve leader peptides encoded by short ORFs often located immediately upstream of antibiotic resistance genes (e.g. Staphylococcus aureus ermA, ermC and cat mRNAs (Bechhofer, 1990; Dreher & Matzura, 1991; Bechhofer, 1993; Drider et al., 2002). In the absence of the antibiotic, the RBS for the resistance determinant is normally sequestered in a secondary structure domain within its cognate mRNA, thus inhibiting translation. However, ribosome stalling induced by subinhibitory concentrations of the antibiotic during translation of the leader peptide results in structural rearrangements that destabilize the inhibitory structure, and therefore allow ribosome binding and subsequent translation of the drug resistance genes, a mechanism known as translation attenuation. The antibiotic-induced stalling in the leader peptide coding sequence highly stabilizes the transcript (Bechhofer & Dubnau, 1987). Thus, ribosome stalling at the 5′-end of an mRNA appears to protect the downstream gene in the same manner as translation initiation complex formation, again suggesting a 5′→3′ directional stabilization, i.e. protection against a 5′-end dependent nuclease activity in Gram-positive organisms.
Kaberdin VR, Bläsi U. Translation initiation and the fate of bacterial mRNAs. FEMS Microbiol Rev. 2006 Nov;30(6):967-79. doi: 10.1111/j.1574-6976.2006.00043.x. Epub 2006 Sep 21. PMID: 16989654.
The ribosomal protein S15 from Escherichia coli represses the translation of its own rpsO mRNA by the entrapment mechanism (Schlax and Worhunsky, 2003) (Figure 1B). In this issue, Marzi et al., 2007 use cryo-electron microscopy (cryo-EM) to provide insights into the structure of the stalled “preinitiation” ribosomal complex containing the structured 5′ UTR region of this mRNA in complex with the repressor protein S15 bound to the platform of the 30S ribosomal subunit (Figure 1B, inset). Interestingly, in this complex, the complementary base pairing interactions are formed between the SD sequence of the mRNA and the small subunit rRNA. Nevertheless, the ribosome is stalled because the start codon is buried in the pseudoknot structure of the mRNA and is thus inaccessible for binding to the initiator tRNA. In the absence of protein S15, the mRNA pseudoknot also engages the ribosome but is then able to unfold, making the start codon accessible. The structure of the stalled initiation complex reveals an extensive network of interactions between the mRNA:S15 complex and numerous ribosomal proteins belonging to the platform and head regions of the small ribosomal subunit.
Marzi et al., 2007 suggest that this structure mimics the initial docking interactions that occur between structured mRNAs and the small ribosomal subunit before SD interactions are established to form a stable initiation complex. These initial interactions between the mRNAs and the small ribosomal subunit have been described on the basis of kinetic and fluorescence energy transfer experiments and termed “stand-by sites” (Studer and Joseph, 2006). (It should be noted that the term “stand-by site” is also used to describe a certain conformation of the SD sequence in the preinitiation state.) It is expected that this initial docking precedes mRNA unfolding, the formation of the SD interactions, and positioning of the start codon. Nevertheless, the stalling interactions between the rpsO mRNA and the ribosome already involve complementary SD interactions, which is generally not the case during the formation of initial stand-by interactions. Based on the structure of the rpsO repressor complex Marzi et al., 2007 compare the binding sites of mRNA sequences in the 5′ UTRs in recent structures of translation initiation complexes of prokaryotes and eukaryotes and propose that a conserved “platform binding center” on the ribosome is formed by ribosomal proteins S2, S7, S11, and RNA helices 26 and 40.
The 30S platform is clearly the foremost binding site of bacterial mRNA in the initiation complex as it harbors the anti-SD sequence that base pairs with the SD sequence of the mRNA. To get an understanding of the whole network of mRNA ribosome interactions during the initiation stage, it is useful to relate the structure obtained by Marzi et al., 2007 to other recent structural data obtained in the field. Crystal structures of the ribosome in complex with the SD sequence oligonucleotides, might resemble the “preinitiation” complex on less structured mRNAs (Yusupova et al., 2006). In these structures the SD-anti-SD helix is positioned in a grove on the 30S platform that includes ribosomal proteins S2, S11, and S18. Furthermore, structures of ribosomes containing mRNA with a poly(U) tail show interactions of the tail with protein S18 in the initiation state (Yusupova et al., 2006). Finally, the structure of the thrS mRNA ribosome complex reveals an mRNA in the final stage of initiation (Jenner et al., 2005). The thrS mRNA harbors a pseudoknot that is recognized by its own translation product threonyl-tRNA synthetase to block the interactions with the small ribosomal subunit by the displacement mechanism (Figure 1A). In this structure the mRNA pseudoknot contacts proteins S11 and S18 and helix 40 of the 16S rRNA. In addition, a three dimensional reconstruction of the bacterial initiation complex assembled in the presence of initiation factors has been obtained by cryo-EM (Allen et al., 2005). In this case Marzi et al. suggests after re-examination of the density reported by Allen and colleagues that an mRNA hairpin located in the 5′ UTR interacts with the platform of the 30S subunit in the vicinity of protein S2.
The analysis of mRNA binding sites can be extended to eukaryotic mRNAs in the special case of translation initiation mediated by internal ribosome entry site (IRES) sequences. In the case of the IRES sequences of hepatitis C virus (HCV) and cricket paralysis virus (CrPV), the binding to the small subunit and positioning of the start codon is mediated exclusively by the mRNA (Boehringer et al., 2005, Schüler et al., 2006). This is in contrast to canonical translation initiation in eukaryotes, in which initiation factors mediate binding of the small ribosomal subunit to the mRNA in the vicinity of the 5′ cap and scanning for the start codon. The cryo-EM structure shows that the HCV IRES is bound to the platform of the 40S subunit including interactions with protein S14e, an S11p homolog, in addition to contacts to the 40S head and body. However, the CrPV IRES is located in the inter-subunit space between the head and body of the 40S subunit. This difference in structure might be due to the different mechanism of translation initiation by the CrPV IRES, which occurs even in the absence of initiation factors and initiator tRNA. The only contacts shared with the HCV IRES outside the mRNA binding cleft occur at protein S5e, an S7p homolog. All these structures highlight the importance of the platform region in the initiation of translation; however, the different nature of the mRNA substrates makes it difficult to put the observed contacts into a unified functional context.
Marzi et al., 2007 point out that conserved patches of amino acids can be found on ribosomal proteins S2, S7 and S11 close to the interaction sites of the rpsO mRNA pseudoknot and many mRNA elements mentioned above. These residues may represent conserved contact points of 5′ UTRs with ribosomes. However, the high degree of conservation is surprising in this context given the diverse nature of mRNA elements interacting with the platform. Conservation of these residues may be required for the interaction with other ribosome associated factors involved in mRNA binding or biogenesis as suggested by recent cryo-EM derived-structures of Era and S1 bound to the ribosome (Sharma et al., 2005).
The results of Marzi et al., 2007 provide exciting structural insight in the initial binding of mRNA to the ribosome during the formation of the initiation complex. These findings illuminate the mechanism of repression of protein synthesis by the S15 protein and further our understanding of the process of translation initiation.
Reference
Boehringer D, Ban N. Trapping the ribosome to control gene expression. Cell. 2007 Sep 21;130(6):983-5. doi: 10.1016/j.cell.2007.09.002. PMID: 17889642.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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