Discussion

Several studies have described quadruplex-mediated alterations in gene expression for eukaryotic systems. Although in computational searches quadruplex-forming sequences have been found enriched in regulatory regions of prokaryotes, studies that systematically investigate their influence in vivo are sparse. In 2012, Chowdhury and co-workers reported the influence of G-quadruplex-stabilizing compounds on the radioresistance of the bacterium Deinococcus radiodurans. As yet, their study is the only one investigating the influence of G-quadruplexes occurring in bacterial promoter regions on gene expression in vivo. However, in their case quadruplexes were enriched in the promoter region of a functional gene class in this respective organism (43). In our studies, we aimed at a more generalized examination by systematically analyzing the influence of G-rich sequences in bacterial gene-regulatory regions. To our knowledge this is the first comprehensive study to show that the effect of G-quadruplexes in the bacterial promoter region on gene expression of the downstream gene is position-dependent. Recently, the influence of strand asymmetry on quadruplex-mediated alteration of transcription was described for eukaryotes by the group of Maiti (301). In their study, a quadruplex sequence in the 5’-UTR only repressed transcription efficiency when placed in the antisense strand. However, translational repression of gene expression was also possible when the G-quadruplex was found in the sense strand (301). The comparison of prokaryotic and eukaryotic systems in this context might prove difficult as genetic mechanisms differ significantly. Hence, conclusions drawn from studies in eukaryotic contexts are not necessarily valid for bacteria; instead separate investigations are necessary.

Our results illustrate that G-quadruplexes can be involved in bacterial gene regulation on both transcriptional and translational levels. We have set up reporter systems based on two different plasmids bearing either eGPF or β-galactosidase reporter genes. First, quadruplex sequences were inserted within the core promoter region (between the unaltered conserved -35 and -10 regions). We found that quadruplex-forming sequences in the core promoter region significantly decreased gene expression by transcriptional modulation when located on the antisense strand, whereas insertion into the sense strand showed much less influence. Introducing different quadruplex sequences into the antisense strand at this position showed that transcriptional repression correlates with quadruplex stability. The non-coding or antisense strand serves as template for the E. coli RNA polymerase. Although the transcription start site is located downstream of the promoter region, polymerase binding to the promoter is essential for transcription initiation. Usually, the E. coli RNA polymerase core enzyme binds to the σ⁷⁰ factor to form the holoenzyme. The sigma factor is responsible for

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promoter recognition. It identifies the -10 and -35 region of the double stranded promoter DNA, forming the closed promoter complex. A σ subunit (σ2) separates the strands of the DNA at the -10 region and binds to the sense (non-template) strand, forming the open promoter complex. Transcription is then initiated by binding of a nucleotide triphosphate (ATP) to the nucleotide +1 at the TSS. An initial transcript of about 10 nucleotides causes the release of the sigma factor from the core RNA polymerase. Finally, the RNA polymerase leaves the promoter region and enters the elongation phase in which the transcription bubble is enlarged to 17 bp and the polymerase moves along the antisense strand (279,302).

Promoter recognition can be strongly influenced by the nucleotide composition of the surrounding 5’-UTR: Upstream elements can enhance the efficiency of RNA polymerase binding. The formation of a secondary structure, like a G-quadruplex, could create a physical barrier that hinders polymerase binding or complicates promoter recognition by σ⁷⁰. In another scenario, polymerase binding could facilitate quadruplex formation, which ultimately might hamper the initiation of transcription or the elongation phase.

In order to investigate the influence of G-rich sequences downstream of the conserved promoter in the 5’-UTR we inserted quadruplexes into the sense and antisense strands 20 nt upstream of the start codon. An increase of gene expression was observed for G-quadruplexes placed into the antisense strand and a repression of gene expression for those inserted in the sense strand. Again, we investigated quadruplexes with different thermodynamic stabilities and observed a correlation between their effect and the quadruplex stability. Compared to controls, mRNA levels remained constant when G-stretches were placed in the sense strand, but were enhanced in constructs bearing the G-sequence in the antisense strand. This points to translational versus transcriptional control of gene expression. The G-quadruplex on the antisense strand influences transcription and might interfere with polymerase elongation or binding. However, the reason for the enhancement of gene expression due to a quadruplex at this position is unclear. A possible explanation could be that E. coli RNA polymerase needs to separate the template and non-template strands for the transcription process. Here, G-quadruplex formation competes with stable G-C Watson-Crick base pairing in the DNA double strand. Formation of a G-quadruplex structure could facilitate strand separation and thereby support the helicase activity of RNA polymerase.

Strands are not separated in the core promoter and the polymerase can bind to the double strand, but in the region downstream of the -10 part the polymerase actively separates the double helix (279,302). This might explain why G-quadruplex, when inserted in the promoter, results in transcriptional repression, whereas its insertion downstream of the promoter increases gene expression. E. coli RNA polymerase has been shown to interact similarly with different promoters (303). Importantly, we observed a similar behavior in two different

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σ⁷⁰-dependent promoters, the constitutive J06 promoter and the araBAD promoter activated by arabinose-induced binding of araC from position -35 to -51 (277,278). However, we cannot exclude the possibility that G-quadruplexes in other systems behave differently.

After having investigated transcriptional regulation by G-quadruplexes in detail in the discussed constructs, we turned to insertions of quadruplexes into transcribed, mRNA-based regions of the 5’-UTR. Especially the initiation of translation seems to be strongly influenced by quadruplex formation, as previously shown in a series of artificially designed, SD-masking expression constructs resulting in down-regulation of translation (161). Masking of the ribosomal binding site is a common mechanism for translational regulation of gene expression, e.g. in riboswitches, RNA thermometers, sRNA-mediated regulation and in artificially designed riboregulators. Different systems have been described using engineered devices as sensitive switches of gene expression in prokaryotic organisms (304-306). In this study, we successfully constructed a system where the SD was masked by means of a hairpin structure. A quadruplex sequence was incorporated into the loop and part of the stem structure so that quadruplex formation destabilized the hairpin structure and thereby liberated the ribosome binding site, effectively resulting in activation of gene expression.

Having shown that quadruplexes located in the vicinity of the SD site are able to strongly influence translation, we wondered whether quadruplexes occur at these positions in natural genetic sequences. Searching the E. coli genome, we found 46 putative G-quadruplexes occurring on the coding strand within the SD region. We investigated the influence of these naturally occurring G-rich stretches within the SD region on gene expression and observed a significant quadruplex-mediated repression for two of the five naturally occurring 5’-UTR regions we investigated. The G-rich region of the 5´-UTR of the oxyR gene was examined in detail and compared to several mutants which should not be able to form a G-quadruplex.

The upstream regions taken from two further genes did not significantly alter gene expression compared to controls. As suggested earlier (161), the secondary structure of a G-quadruplex might complicate the binding of the ribosome to the SD region and thereby decrease gene expression efficiency. However, for the quadruplex near the SD site occurring in the napH 5’-UTR we observed an increase of gene expression compared to two controls.

As described earlier, the overall 5’-UTR nucleotide composition has a strong impact on translation (284). Also, in our engineered system, the sequence surrounding the SD region plays an important role in quadruplex-mediated translational modification. Competing secondary structures can be dissolved by quadruplexes and thus enable the access to the SD region. As we have no concrete knowledge of the exact sequence of the SD region in this case, it is difficult to draw mechanistic conclusions.

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We also tested the influence of the quadruplex stabilizing compound NMM, but no effects on gene expression were observed in our systems. Although the use of quadruplex-stabilizing compounds is common in this field, in vivo most of them have been applied to eukaryotic cells (e.g. yeast (15)). Regarding bacteria, NMM was reported to stabilize G-quadruplex structures in Neisseria gonorrhoeae (44) and Deinococcus radiodurans (43). However, the effects of NMM on G-quadruplexes in E. coli in particular have not been shown so far. Also, the addition of other compounds (TMPyP4, 360A) did not lead to an intensifying effect on gene expression changes. It is possible that those compounds are not properly absorbed in E. coli species – prokaryotic uptake mechanisms of said compounds have not been described so far. Furthermore, there is no information on intracellular degradation and half-lives. Consequently, the incubation time of the compound might prove critical as well.

Potentially, as fluorescence and luminescence molecules have long half-lives our read-out systems may not be suitable for detecting an enhanced effect in gene expression changes.

So far reporter systems used in combination with quadruplex-binding ligands in prokaryotes differed from ours and were also more sensitive (radioactivity (43), phase-variation assay (44)).

Finally, we showed that G-quadruplex sequences inserted 4 nt after the stop codon of a reporter gene did not result in a consistent modulation of gene expression. The findings about G-quadruplex functions in regulatory regions are summarized in Figure 3.14.

Figure 3.14: Summary of effects mediated by G-quadruplexes in regulatory regions.

Different quadruplex insertion sites and the respective effects are shown. Red arrows pointing up: increased gene expression; red arrows pointing down: decreased gene expression. Dashed red line indicates the sequence range which was modified for investigation of the SD adjacent region.

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Apart from G-quadruplexes in regulatory regions, we also conducted first experiments investigating G-quadruplexes within bacterial ORFs. Sugimoto and co-workers published a series of experiments that showed G-quadruplexes at these positions to be responsible for ribosomal halt, frameshifting or the production of a truncated product. Although they did in vitro studies with G-quadruplex sequences found in the E. coli ORFs, all in vivo studies were performed with eukaryotic cells. The aim of our investigations was to allocate an in vivo function to quadruplexes within bacterial ORFs. We found two different potential G-quadruplex motifs present in K⁺ transporters of Salmonella subspecies. After in vitro characterization we decided to design constructs that express these genes with the potential G-quadruplexes (or mutated versions) and investigate them via Western blotting. However, our results were inconclusive: We did neither observe the formation of a truncated or elongated protein product nor consistent enhancement or repression of protein expression.

Although our in vivo investigations were conducted in a bacterial system, we investigated a sequence derived from Salmonella in E. coli. E. coli and Salmonella enterica species can be considered phylogenetically related (307) and share a large amount of their genomic material (308,309), however, they display lifestyle divergences (310,311) and it can only be assumed that gene regulatory mechanisms are similar. Also, in Salmonella subspecies specific proteins could exist that recognize quadruplex structures, but are not present in E. coli. To exclude organism-specific effects in vivo analysis in Salmonella should be conducted.

Nevertheless, we have only just begun the investigations in this field, and there are lots of possibilities to improve the experimental procedures. Western blot analysis is not only very time-intensive, but also has a rather low sensitivity, potentially making it impossible to detect small portions of truncated or elongated proteins. The design of an artificial system where the potential G-quadruplex would occur within a read-out gene (e.g. eGFP) would be an option to facilitate the analysis and to test different G-quadruplex sequences for their function within ORFs. One could also try to intensify the G-quadruplex-mediated effect by stabilizing the structure with G-quadruplex-interacting compounds. First hints as to the function in bacteria could also arise from an in vitro translation assay.

In conclusion, the comprehensive study presented here gives new insights into quadruplex-mediated regulation of gene expression in E. coli. We were able to show that both the strand orientation and the exact position of a G-quadruplex in the 5’-UTR strongly influence its effect on transcription. Translational alterations are also dependent on the position and the surrounding sequence of the G-quadruplex in the 5’-UTR. Although the presented data do not show a direct role of natural quadruplexes in gene regulation, we cannot exclude the possibility of quadruplexes playing functional roles in controlling gene expression. In such a scenario it might be possible that these distinct structures are specifically induced under

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certain conditions. It is important to note that intracellular K⁺ concentrations increase in response to osmotic upshock and environmental stresses (270,312). Intriguingly, G-quadruplexes are stabilized by monovalent ions showing the highest affinity for K⁺. We found several G-quadruplexes in the 5’-UTRs of genes related to stress responses (see Table 13.1 in the appendices). OxyR, the oxidative stress regulator, is a transcriptional regulator in the oxidative and nitrosative stress response (313,314). RelA encodes for an enzyme involved in the stringent response which activates the synthesis of the regulatory molecules Guanosine-3’-5’-bis(diphosphate) (ppGpp) and Guanosine-3’-diphosphate-5’-triphosphate (pppGpp), both acting as alarmones to amino acid starvation (315). RseA can inhibit and regulate the sigma E factor (316). The Sigma E system is involved in the responses to heat shock, osmotic stress or other stresses on membrane and periplasmic proteins. Other genes related to environmental stress have been identified in our search (see Table 13.1 in the appendices). Interestingly, also the genes found in within the ORFs of Salmonella species were related to the K⁺ transporter (kdpD) expressed upon osmotic stress. One could speculate that the identified quadruplex motifs might function as regulatory units responding to stress or other environmental changes. However, stress responses and bacterial lifestyle changes are regulated by several complex and overlaying pathways. This makes it difficult to prove the formation and influence of a quadruplex structure which, in addition, might only form temporarily. We recently carried out initial experiments with osmotic up-shock that should have resulted in temporarily increased intracellular K⁺ levels, but found no conclusive influence in reporter gene assays (data not shown). However, further experiments along these lines utilizing even more suited reporter gene assays might be able to shed more light on the possibility of quadruplex formation as a natural mechanism for conditional gene regulation. Also, one should keep in mind that the presence of a G-quadruplex motif in one strand is inevitably tied to the presence of a C-rich pattern, the so called i-motif, in the complementary strand. Although the formation of i-motifs is only reported for lower pH (317), it is possible that they might hold a functional role as well.

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