Binding of 30S Ribosome Induces Single-stranded Conformation Within and Downstream of the Expression Platform in a Translational Riboswitch

Translational riboswitches are bacterial gene regulatory elements found in the 5′-untranslated region of mRNAs. They operate through a conformational refolding reaction that is triggered by a concentration change of a modulating small molecular ligand. The translation initiation region (TIR) is either released from or incorporated into base pairing interactions through the conformational switch. Hence, initiation of translation is regulated by the accessibility of the Shine-Dalgarno sequence and start codon. Interaction with the 30S ribosome is indispensable for the structural switch between functional OFF and ON states. However, on a molecular level it is still not fully resolved how the ribosome is accommodated near or at the translation initiation region in the context of translational riboswitches. The standby model of translation initiation postulates a binding site where the mRNA enters the ribosome and where it resides until the initiation site becomes unstructured and accessible. We here investigated the adenine-sensing riboswitch from Vibrio vulnificus. By application of a ¹⁹F labelling strategy for NMR spectroscopy that utilizes ligation techniques to synthesize differentially ¹⁹F labelled riboswitch molecules we show that nucleotides directly downstream of the riboswitch domain are first involved in productive interaction with the 30S ribosomal subunit. Upon the concerted action of ligand and the ribosomal protein rS1 the TIR becomes available and subsequently the 30S ribosome can slide towards the TIR. It will be interesting to see whether this is a general feature in translational riboswitches or if riboswitches exist where this region is structured and represent yet another layer of regulation.     

Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA

The 16S rRNA-binding ribosomal protein S15 is a key component in the assembly of the small ribosomal subunit in bacteria. We have shown that S15 from the extreme thermophile Thermus thermophilus represses the translation of its own mRNA in vitro, by interacting with the leader segment of its mRNA. The S15 mRNA-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis. S15 binding triggers a conformational rearrangement of its mRNA into a fold that mimics the conserved three-way junction of the S15 rRNA-binding site. This conformational change masks the ribosome entry site, as demonstrated by direct competition between the ribosomal subunit and S15 for mRNA binding. A comparison of the T.thermophilus and Escherichia coli regulation systems reveals that the two regulatory mRNA targets do not share any similarity and that the mechanisms of translational inhibition are different. Our results highlight an astonishing plasticity of mRNA in its ability to adapt to evolutionary constraints, that contrasts with the extreme conservation of the rRNA-binding site.     

Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA

Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon. In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) [Tang, C. K., & Draper, D. E. (1989) Cell 57, 531]. We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced. Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured. Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression. An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly. Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding. We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled. Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding. This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.     

Quantitative correlation between mRNA secondary structure around the region downstream of the initiation codon and translational efficiency in Escherichia coli

Translational efficiency in Escherichia coli is known to be strongly influenced by the secondary structure around the ribosome‐binding site and the initiation codon in the translational‐initiation region of the mRNA. Several quantitative studies have reported that translational efficiency is attributable to effects on ribosome accessibility predominantly caused by the secondary structure surrounding the ribosome‐binding site. However, the influence of mRNA secondary structure around regions downstream of the initiation codon on translational efficiency after ribosome‐binding step has not been quantitatively studied. Here, we quantitatively analyzed the relationship between secondary structure of mRNA surrounding the region downstream of the initiation codon, referred to as the downstream region (DR), and protein expression levels. Modified hairpin structures containing the initiation codon were constructed by site‐directed mutagenesis, and their effects on expression were analyzed in vivo. The minimal folding free energy (ΔG) of a local hairpin structure was found to be linearly correlated with the relative expression level over a range of fourfold change. These results demonstrate that expression level can be quantitatively controlled by changing the stability of the secondary structure surrounding the DR. Biotechnol. Bioeng. 2009; 104: 611–616 © 2009 Wiley Periodicals, Inc.     

The dependence of the E.coli gene expression level on the structure of the translation initiation region (TIR). IV. Distal complementary TIR interactions with the mRNA coding region

To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA. A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation. The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.     

Autoregulation of gene expression: Quantitative evaluation of the expression and function of the bacteriophage T4 gene 32 (single-stranded DNA binding) protein system

The free concentration of bacteriophage T4-coded gene 32 (single-stranded DNA binding) protein in the cell is autoregulated at the translational level during T4 infection of Escherichia coli. The control of the synthesis of this protein reflects the following progression of net (co-operative) binding affinities for the various potential nucleic acid binding targets present: single-stranded DNA > gene 32 mRNA > other T4 mRNAs ? double-stranded DNA. In this paper we show that the free concentration of gene 32 protein is maintained at 2 to 3 μm, and use the measured binding parameters for gene 32 protein, extrapolated to intracellular conditions, to provide a quantitative molecular interpretation of this system of control of gene expression. These results are then further utilized to define the specific autoregulatory binding sequence (translational operator site) on the gene 32 mRNA as a uniquely unstructured finite binding lattice terminated by elements of secondary structure not subject to melting by gene 32 protein at the autoregulated concentration, and to predict how this site must differ from those found on other T4 messenger RNAs. It is shown that these predictions are fully consistent with available T4 DNA sequence data. The control of free protein concentration as a method of genome regulation is discussed in terms of other systems to which these approaches may apply.     

Independent and coupled translational initiation of atp genes in Escherichia coli: experiments using chromosomal and plasmid-borne lacZ fusions

The translational initiation rates directed by the translational initiation regions (TIRs) of the atpB, atpH, atpA and atpG genes of Escherichia coli were investigated using lacZ fusions present on plasmids as well as integrated into the chromosome. This was the first investigation of the translational efficiency of the atpB gene, whose unfused product (subunit a) can be toxic to the cell. The specific mRNA levels, rates of in vivo protein synthesis and beta-galactosidase activities encoded by the atp::lacZ fusions were compared in order to obtain valid estimates of relative translation rates. The results indicate that in the E. coli atp operon, translation directed by the atpB, atpH and atpG TIRs is less efficient than that directed by the atpA TIR, and are thus consistent with earlier measurements of direct atp gene expression. Initiation is, however, to differing extents, controlled by coupling to the translation of upstream neighbours. There is particularly tight coupling between atpH and atpA. Increasing the distance between these two genes whilst maintaining the original atpA TIR structure decreased the degree of coupling. The influence of manipulations of the atpG TIR structure upon translational efficiency was quantitatively more pronounced when the atpG fusions were present as a single copy per chromosome. This is likely to be related to the mRNA binding characteristics of 30S ribosomal subunits and/or to the influence of other (trans-acting) factors. The control of independent and coupled initiation at the atp TIRs is discussed in relation to mRNA structure and possible cis and trans regulatory phenomena.     

Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region

Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine- Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message- specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.     

The role of bases upstream of the Shine-Dalgarno region and in the coding sequence in the control of gene expression in Escherichia coli: translation and stability of mRNAs in vivo

A range of translational initiation regions (TIR) was created by combining synthetic DNA fragments derived from the atpB-atpE intercistronic sequence of Escherichia coli with the cDNA sequence encoding mature human interleukin 2 (IL-2), the E. coli fnr gene, or an fnr::lacZ gene fusion. Both the overall rates of gene expression and the relative concentrations and stabilities of the corresponding mRNA species were estimated in strains bearing the constructs on plasmids. These measurements served as the basis for analyses of the relationship between the structure of the TIR and the true rates of translation that it promotes. The constructs involving the IL-2 cDNA were predicted to allow much less stable secondary structure within the TIR than those involving the N-terminal region of the fnr gene. Thus by combining one set of upstream sequences with two different types of N-terminal coding sequence, it was possible to distinguish between the respective influences of primary and secondary structure upon initiation. The data indicate that in the presence of a given Shine-Dalgarno (SD)/start codon combination, the decisive factor for translational initiation efficiency is the stability of base pairing involving, or in the vicinity of, this region. The sequences contributing to this secondary structure can be many bases upstream of the SD region and/or downstream of the start codon. There was no indication that the specific base sequence upstream of the SD region could, other than to the extent that it contributed to the local secondary structure, significantly influence the efficiency of translational initiation.     

An unstructured mRNA region and a 5' hairpin represent important elements of the E. coli translation initiation signal determined by using the bacteriophage T7 gene 1 translation start site.

Gene 1 of bacteriophage T7 early region--the RNA polymerase gene--is very actively translated during the infectious cycle of this phage. A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr). Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity. Computer analysis revealed a potentially inhibitory hairpin binding the S-D sequence into its stem base paired to vector-derived upstream sequences. Mutational alterations demonstrated that this hairpin was not responsible for the low activity. However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude. Computer analysis of this sequence, including nucleotide shuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5' end, here formed solely by T7 sequences. There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin. Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies. The delta G values of these regions were higher, i.e. potential secondary structural elements were fewer, than in TIR of genes from E. coli. This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes. When the 5' ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region. Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence. We suggest that the formation of this 5' hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR. In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude.     

An antisense RNA inhibits translation by competing with standby ribosomes

Most antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an antisense RNA inhibits translation without binding directly to a TIR. The tisAB locus encodes an SOS-induced toxin, and IstR-1 is the antisense RNA that counteracts toxicity. We show that full-length tisAB mRNA (+1) is translationally inactive and endonucleolytic processing produces an active mRNA (+42). IstR-1 binding inhibits translation of this mRNA, and subsequent RNase III cleavage generates a truncated, inactive mRNA (+106). In vitro translation, toeprinting, and structure mapping suggest that active, but not inactive, tisAB mRNAs contain an upstream ribosome loading or "standby" site. Standby binding is required for initiation at the highly structured tisB TIR. This may involve ribosome sliding to a transiently open tisB TIR. IstR-1 competes with ribosomes by base pairing to the standby site located approximately 100 nucleotides upstream.