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# 5'UTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability

Fan Chen, Muriel Cocaign-Bousquet, Laurence Girbal, Sébastien Nouaille

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Fan Chen, Muriel Cocaign-Bousquet, Laurence Girbal, Sébastien Nouaille. 5'UTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability. Frontiers in Microbiology, 2022, 13, pp.1088941. 10.3389/fmicb.2022.1088941. hal-04012701

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EDITED BY

Stefano Marzi,
UPR9002 Architecture et
Réactivité de l'arN, France

REVIEWED BY

Franz Narberhaus,
Ruhr University Bochum,
Germany
Harald Putzer,
UMR8261 Expression Génétique
Microbienne (EGM), France

*CORRESPONDENCE

Muriel Cocaign-Bousquet

✉ cocaign@insa-tonouse.fr

Sébastien Nouaille

✉ sebastien.nouaille@insa-tonouse.fr

'These authors share senior authorship and
last authorship

SPECIALTY SECTION

This article was submitted to
Microbial Physiology and Metabolism,
a section of the journal
Frontiers in Microbiology

RECEIVED 03 November 2022

ACCEPTED 02 December 2022

PUBLISHED 21 December 2022

CITATION

Chen F, Cocaign-Bousquet M, Girbal L and
Nouaille S (2022) 5'UTR sequences
influence protein levels in *Escherichia coli*
by regulating translation initiation and
mRNA stability.
*Front. Microbiol.* 13:1088941.
doi: 10.3389/fmicb.2022.1088941

COPYRIGHT

© 2022 Chen, Cocaign-Bousquet, Girbal and Nouaille. This is an open-access article distributed under the terms of the [Creative Commons Attribution License (CC BY)](https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

5'UTR sequences influence
protein levels in *Escherichia coli*
by regulating translation
initiation and mRNA stability

Fan Chen, Muriel Cocaign-Bousquet*, Laurence Girbal† and Sébastien Nouaille*†

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France

A set of 41 synthetic 5'UTRs with different theoretical translation initiation rates were generated to explore the role of 5'UTRs in the regulation of protein levels in *Escherichia coli*. The roles of the synthetic 5'UTRs in regulating the expression of different reporter genes were analyzed *in vivo*. Protein levels varied substantially between the different constructs but for most of the 5'UTRs, protein levels were not correlated with theoretical translation initiation rates. Large variations in mRNA concentrations were measured with the different 5'UTRs even though the same concentration of transcription inducer was used in each case. 5'UTRs were also found to strongly affect mRNA stability, and these changes in mRNA stability often contributed to observed differences in mRNA concentration. Unexpectedly, the effect of the 5'UTRs on mRNA half-lives was found to vary depending on the downstream reporter gene. These results clearly demonstrate that 5'UTRs contribute to gene expression regulation at the level of translation initiation and of mRNA stability, to an extent that depends on the nature of the downstream gene.

KEYWORDS

mRNA stability, translation initiation, post-transcriptional regulation, gene expression regulation, protein level regulation, *Escherichia coli*

Introduction

*Escherichia coli* cells adapt their metabolism to changing environments by modifying the expression pattern of their gene repertoire. Gene expression control encompasses a wide range of transcriptional and post-transcriptional mechanisms that affect different stages of gene expression.

The 5'UTR of mRNA, the transcribed but untranslated region from the transcription start site to the first nucleotide of the translational start codon, is a potential regulatory element for gene expression. 5'UTRs are crucial for efficient translation. Their most important features are generally considered to be the ribosome binding site, the Shine-Dalgarno (SD) sequence, complementary to the anti-SD sequence at the 3'end of 16S rRNA (Shine and Dalgarno, 1974), and A/U richness, which limits the formation of
secondary structural elements in the ribosome binding site (Komarova et al., 2002; Kozak, 2005). Strong SD/anti-SD pairing and weak secondary structure in 5'UTRs are the hallmarks of highly expressed genes in *E. coli* (Prabhakaran et al., 2015). The regions upstream of the SD sequence and the distance between the SD sequence and the initiation codon also play important roles (Hartz et al., 1991; Komarova et al., 2002; Osterman et al., 2013). Translation initiation of particular mRNAs is also affected by 5'UTR binding of regulatory intracellular molecules, metabolites in the case of riboswitches (Tucker and Breaker, 2005), small RNA (sRNA) molecules (Gottesman and Storz, 2011; Lalaouna et al., 2013; Wagner and Romby, 2015) and RNA-binding proteins (CsrA, Hfq, and ProQ; Folichon et al., 2003; Holmqvist et al., 2018; Holmqvist and Vogel, 2018; Romeo and Babitzke, 2018).

The effect of 5'UTRs on translation indirectly influences mRNA stability (Iost and Dreyfus, 1995; Komarova et al., 2005) because limiting translation initiation destabilizes mRNA. Mutating the ribosome binding site of the *lacZ* reporter gene has been shown to reduce the efficiency of ribosome binding, leading to a decrease in *lacZ* mRNA stability (Yarchuk et al., 1992; Iost and Dreyfus, 1995). The structured motifs of 5'UTRs also modulate the efficiency of translation initiation by sequestering/exposing the SD sequence, thereby reducing/increasing its accessibility to ribosomes (Chowdhury et al., 2006). The protective effect of high ribosomal occupancy is thought to arise from ribosomes outcompeting RNase E, thereby preventing 5'-end-dependent degradation of the transcript (Dreyfus, 2009). mRNA stability is also directly regulated by the binding of sRNAs and RNA binding proteins to 5'UTRs, which can activate or prevent RNase E scanning and cleavage (Ikeda et al., 2011; Prevost et al., 2011; Vakulskas et al., 2015; Richards and Belasco, 2019).

In this study, to further investigate the role of 5'UTR sequences in gene expression regulation in *E. coli*, we measured the effects of a large set of synthetic 5'UTR sequences on mRNA and protein levels and mRNA stability. The synthetic 5'UTR sequences were chosen to cover a large range of theoretical translation initiation rates and were placed under the control of an inducible promoter and fused to different reporter genes. This approach has several advantages. (i) The use of synthetic 5'UTRs instead of native sequences limits the risk of interference with the binding of intracellular regulatory molecules. (ii) Length effects are avoided because the considered 5'UTRs are identical in length. (iii) No knowledge of specific regulatory elements within the 5'UTR sequence is required because the entire 5'UTR sequence is replaced in each case. (iv) Similar levels of transcription initiation can be applied to reduce differences in mRNA synthesis between the constructs. And (v), the interplay between the effects of the 5'UTRs and of the downstream gene can be explored. Results show that for most of the synthetic 5'UTRs, the theoretical rate of translation initiation is not a good predictor of protein levels. Many 5'UTRs were found to regulate mRNA levels by altering the stability of the transcript in a downstream gene dependent manner. Overall, our results demonstrate that 5'UTRs act as

“regulatory-hubs” of gene expression in close interaction with the downstream gene.

# Materials and methods

## Design of synthetic 5'UTR sequences

RBS Calculator was used to design synthetic 5'UTR sequences with different translation initiation rates (Salis, 2011). RBS Calculator uses a statistical thermodynamic model considering Gibbs free energies for key molecular interactions in translation initiation to give an estimation of translation initiation rate. RBS Calculator was used to generate synthetic 5'UTR sequences (containing a RBS) with rationally controlled translation initiation rates over a 100,000-fold range. The 5'UTRs were designed based on the first 150 nucleotides of the *lacZ* coding sequence. The reference 5'UTR was taken from the $P_{BAD}$-*lacZ* control plasmid from the Invitrogen $P_{BAD}$-his/myc expression system used for the production of heterologous proteins in *E. coli*. This 5'UTR is 33 nt long with an efficient SD sequence (GGAGG) and an RBS index of 33,000 arbitrary units (AU). Ten RBS indexes were targeted to cover a wide range of values (100, 250, 500, 1,000, 1,500, 2,500, 5,000, 33,000, 66,000, and 100,000 AU). For each RBS index, 200 unique 33 nt 5'UTR sequences were generated by RBS Calculator and from 3 to 6 sequences were manually selected from each class, with high GC% and with low GC%. One additional criterion was that the sequences should not have a strong folding propensity (minimum free energy calculated using the mfold software) within the 5'UTR sequence or at the beginning of the *lacZ* coding sequence. Including the reference sequence, this procedure yielded a set of 41 synthetic 5'UTR sequences covering a wide range of RBS indexes (Supplementary Table S1).

## Plasmid constructions

The synthetic 5'UTRs were introduced in place of the reference 5'UTR in the $P_{BAD}$-*lacZ* plasmid by PCR. For each 5'UTR, a primer was designed to hybridize with the recipient vector at the ATG of the *lacZ* CDS at the 3' end and was extended with the 5'UTR specifying sequence at the 5'end. These were used in combination with a reverse primer hybridizing to the promoter region from the nucleotide upstream of the +1 transcription start site. The full plasmid was amplified by PCR using *Phusion* polymerase (New England Biolabs). The amplicons were gel-purified and the 5'ends were phosphorylated with T4 polynucleotide kinase (New England Biolabs) and self-ligated with T4 DNA ligase. The ligation mix was used to transform *E. coli* DH5α cells (New England Biolabs). The primers used for 5'UTR cloning are listed in Supplementary Table S2. Plasmid DNA was isolated using the QIAprep Miniprep kit (Qiagen) and constructs were verified by sequencing (Eurofins). To replace the reporter gene, the CDS from *msfGFP* (encoding monomeric superfolder
green fluorescent protein) and *txAbF* (encoding an α-L-arabinofuranosidase from *Thermobacillus xylanilyticus*) were amplified by PCR. The whole plasmid backbones containing the eight selected 5'UTRs fused to *lacZ* were PCR amplified from the end of the *lacZ* coding sequence to the 5'UTR. The amplicons were gel purified and the *msfGFP* or *txAbF* CDS inserts were phosphorylated, ligated with the backbones, and transferred to *E. coli* DH5α cells. The fusions between the 5'UTRs and the reporter genes were verified by sequencing.

## Bacterial strains, growth, and induction conditions

All cloning steps were performed using *E. coli* DH5α cells (New England Biolabs). Cell cultures were grown in LB or LB-agar. All constructs were transferred into the reference strain (MET 345) for characterization. MET 345 is a derivative strain of DLT 2202 (MG1655 Δ*araFGH*, Ω*pcp18::araE533*) lacking the chromosomal copy of *lacZ* (Ah-Seng et al., 2013; Nouaille et al., 2017). In this strain, the PBAD promoter is proportionally induced by the concentration of arabinose uniformly in all cells. All the strains were routinely grown in M9 minimal medium (Esquerre et al., 2014) supplemented with ampicillin 100 µg/ml at 37°C, 150 rpm unless otherwise stated. Cultures were inoculated from overnight precultures at an initial OD₆₀₀m of 0.1. Induction levels were evaluated by adding arabinose to exponentially growing cultures (OD₆₀₀ = 0.6) with serial dilutions for final concentrations of 0.00001 to 0.1% arabinose. For physiological characterizations, strains carrying *lacZ* and *txAbF* were induced with 0.001% arabinose, and strains carrying *msfGFP* were induced with 0.01% arabinose. Samples were taken 30 min after arabinose induction.

## Measurements of β-galactosidase and α-L-arabinofuranosidase activity

Harvested cells (3 mg dry weight) were washed twice with cold 0.2% KCl, resuspended in 1 ml of breaking buffer (15 mM Tris 400 mM/ tricarballylate 1, 4.5% Glycerol, 0.9 mM MgCl2, 0.2 mM DTT; pH = 7.2) and transferred into screw capped tubes containing 0.1 g of glass beads. The cells were disrupted with a FastPrep-24 instrument (MP Biomedicals) in six cycles (6.5 m/s, 30 s) with 1 min on ice between each cycle. After centrifugation, measurements were performed on the supernatants containing soluble proteins. All measurements were performed in triplicate on three biological replicates. β-Galactosidase activity was determined colorimetrically using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a substrate (Miller, 1972). α-L-arabinofuranosidase activity was measured using discontinuous assays, a colorimetric method (Bissaro et al., 2014) based on 4-Nitrophenyl-α-L-arabinofuranoside (pNP-Araf, colorless), which is hydrolyzed by α-L-arabinofuranosidase and releases p-nitrophenyl (pNP, yellow). Samples (100 µL) were

diluted in sodium phosphate (100 mM) and placed in 96-well microplates. Assays were initiated by adding 100 µL of 2X assay buffer. The assay buffer (1×) consisted of 100 mM sodium phosphate at pH 7, 1 mM MgCl₂, 50 mM β-mercaptoethanol and 10 mM pNP-Araf. Absorbance was measured at 401 nm using a microplate reader (SpectraMax Plus 384). Kinetic readings began immediately after the assay was initiated by adding 2x assay buffer, with absorbance measurements every 20s for 30 min at 45°C. α-L-arabinofuranosidase activity was measured in units defined by the catalysis of 1 µmol of pNP per minute. Total protein was assayed using the Bradford method with bovine serum albumin as the protein standard (Bradford, 1976).

## Fluorescence measurements

Specific fluorescence was defined as relative fluorescence divided by the corresponding OD₆₀₀m. Strains carrying the *msfGFP* gene were grown and inducted as described above. One hundred microliter cultures were serially diluted 1:2 with cold M9 medium four times on a microplate on ice (final volume, 300 µL). Fluorescence and cell density (OD₆₀₀m) measurements were then performed using a Bio TeK Synergy H1 microplate reader (BioTek, Winooski, United States). The reference plasmid-less strain MET 345 was used as a negative control and the corresponding fluorescence intensity was subtracted as background. The excitation and emission wavelengths of msfGFP are 475 and 510 nm. Measurements were performed in triplicate on three biological replicates.

## mRNA concentration and half-life determination

Cells (3 mg dry weight) were harvested and flash frozen in liquid nitrogen, before rifampicin addition (reference point, T0) and 0.3, 0.6, 1, 1.5, 2, 3, 4, 5, 6, 7.5, 10, 12.5, and 15 min after adding rifampicin (500 mg/l) to arrest transcription initiation. Samples were stored at -80°C until mRNA extraction. Stability measurements were performed on six samples (T0 plus five samples extracted after rifampicin addition). mRNA was extracted using the RNeasy Mini extraction kit (Qiagen) according to manufacturer instructions. Briefly, after thawing on ice, samples were centrifuged and the cell pellet was resuspended in 500 µL of RLT buffer supplemented with 0.01% β-mercaptoethanol (Sigma) and the mixture was transferred into a tube containing glass beads (0.1 g). The cells were disrupted with a FastPrep-24 instrument (MP Biomedicals) in three 30 s cycles at 6.5 m/s, with 1 min on ice between each cycle, and centrifuged for 10 min at 13,200 g at 4°C. Any DNA contamination was eliminated using the TURBO DNA-freeTM Kit (Ambion) according to manufacturer instructions. DNase-treated RNA was then quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific) and the integrity of the RNA was verified using a 2,100 Bioanalyzer
(Agilent) with the RNA 6000 Nano LabChip kit (Agilent). The RNA was stored at -80°C until required.

For cDNA synthesis, 5 µg of total RNA was mixed with 1 µL of random primers (500 ng/µL; Life Technologies) and RNase-free water to a final volume of 24 µL. The mixture was heated at 70°C for 5 min and then immediately cooled to 4°C. SuperScript II reverse transcriptase (200 units) was then added with 0.5 M DTT, 15 mM dNTP Mix, and 5x first strand buffer (Life Technologies). Reverse transcription was performed at 42°C for 1 h, followed by inactivation at 70°C for 15 min. The RNA-cDNA hybrids were then degraded by adding 5 U of RNase H (Life Technologies). The cDNA was further purified using Illustra Microspin G-25 columns (GE Healthcare) according to manufacturer instructions.

Quantitative RT-PCR was performed using SYBR Green Supermix (Bio-Rad) on a LightCycler 480 II thermal cycler system (Roche, Mannheim, Germany) with the following temperature program: 5 min at 95°C for pre-incubation; then 45 PCR cycles 10 s at 95°C for denaturation, 10 s at 60°C for annealing, and 10 s at 72°C for elongation. The melting curve analysis protocol consisted of 1 min at 65°C followed by heating to 95°C at a rate of 1°C every 9 s. The qRT-PCR measurements of mRNA stability were performed at the Gentiane platform (INRAE, Clermont Ferrand, France) using the high-throughput Fluidigm method (Biomark). LightCycler quantifications were performed on at least duplicates of each sample combined with at least duplicates of each primer pair. mRNA concentration measurements with the Fluidigm method were performed on triplicates of each cDNA sample combined with 2 to 5 technical replicates of each primer pair. mRNA half-lives were measured in triplicate for each primer pair on one biological replicate.

mRNA concentration measurements by qRT-PCR were performed using the Pfaffl method (Pfaffl, 2001). After normalization to the fold change of the reference gene (ihfB, integration host factor β-subunit), results were expressed as the mean n-fold difference (± standard deviation) between the tested strain and the reference strain (MET 346, 5'UTR_33k_30-reporter). The half-lives ($t_{1/2}$) of the mRNA species were obtained from the degradation rates ($k, t_{1/2} = 1/k$) after rifampicin addition. The degradation rates were obtained by linear regression of the time evolutions of the cycle threshold (Ct) after rifampicin addition. Since Ct values are very sensitive to small changes in concentration, no attempt was made to estimate delays in transcript degradation after rifampicin addition as this would have been too uncertain. The half-lives were therefore calculated from the linear regression coefficients (the degradation rates, $k$) of the Ct versus time curves provided the fits had $R^2 > 0.85$.

## Primer design

Primers for qPCR were designed using Vector NTI Advance 11 (Life Technologies) with a melting temperature of 59-61°C, a length of 20-22 bp and 50-67% GC content as constraints, leading to amplicons ranging from 75 to 148 bp in size. The reaction

efficiency of each pair of primers was tested as a single amplicon on serial dilutions of *lacZ*, *txAbF*, or *msfGFP*-containing plasmid, depending on the primer pairs analyzed. Primer pairs were validated if the qPCR efficiency was between 90 and 110% over the dilution range tested. For each reporter gene (*lacZ*, *txAbF*, and *msfGFP*), three primer pairs were designed and distributed equally along the CDSs (beginning, middle, and end of the sequence). The housekeeping gene *ihfB* was used as an internal normalization control.

## Degradational regulation coefficient

The degradational regulation coefficient (pD) represents the contribution of stability regulation in the control of mRNA concentration (Esquerre et al., 2014). Comparing pD between two strains with different mRNA concentrations shows if the difference in mRNA concentration is due to a difference in stability. Assuming that a steady state has been reached, differences in pD between two strains (the strain of interest and the reference strain) can be calculated as the negative value of the slope of the double-logarithmic plot of the degradation rate constant ($k$) against the initial mRNA concentration (before rifampicin treatment) in the two compared strains, $pD = -dlnk/(dln[mRNA])$ where dlnk and dln[mRNA] are, respectively, the differences between the logarithmic degradation rate constants and mRNA concentrations between the strain of interest and the reference strain.

## Results

### Effect of 5'UTR sequences on β-galactosidase protein activity

We first designed a set of 41 synthetic 5'UTR sequences, 33 nt long, which in combination with the first 150 nt of *lacZ* cover a wide range of RBS indexes. These were cloned upstream of the *lacZ* reporter gene under transcriptional control of the P<sub>BAD</sub> promoter and the constructs were transferred into an *E. coli* MG1655 derivative strain lacking the chromosomal copy of *lacZ*. We then measured specific β-galactosidase activity (as a good proxy for the β-galactosidase protein level) of the 41 strains after 30 min of induction with arabinose during exponential growth on synthetic glucose medium. Specific β-galactosidase activity varied widely between strains, barely above the detection threshold in some strains, while in other strains, the activity ranged from 0.01 ± 0.00 to 12.14 ± 1.40 µmol/min/mg of protein (Figure 1). The highest and lowest β-galactosidase activities corresponded, respectively, to the highest and lowest RBS indexes (5'UTR_100k_41 and 5'UTR_100_1), but for most of the 5'UTRs, protein activity was not correlated with the theoretical rate of translation initiation. Some 5'UTRs with a high RBS index (e.g., 5'UTR_100k_38) yielded low protein activities, while other 5'UTRs with a low RBS index (such as 5'UTR_2,500_18) yielded high protein activities.
<figure><img src="image_4.png" /><figcaption>FIGURE 1</figcaption></figure>

β-galactosidase activity associated with 41 synthetic 5'UTRs ordered by RBS index from 100 to 100,000AU. Each cluster of columns with the same color corresponds to a specific RBS index series. Error bars represent standard deviations (n = 9).

Furthermore, several 5'UTRs with the same theoretical translation initiation rate (such as the 5'UTR-33 k series with RBS indexes around 33,000) led to different protein activities (25-fold difference between the lowest and the highest producer of the 33 k series). Altogether, these results show that for most of the 5'UTRs in this experimental model, the theoretical translation initiation rate was not a good predictor of protein activity and by approximation neither of its concentration. This implies that 5'UTR-mediated regulation of translation initiation is not the only determinant of protein level and that additional 5'UTR-associated factors must be involved. We therefore examined the possibility of 5'UTR-associated changes in *lacZ* mRNA concentration.

## Effect of 5'UTR sequences on *lacZ* mRNA levels

The *lacZ* mRNA concentration was found to vary considerably between the 41 strains with different synthetic 5'UTRs (Figure 2; ~300-fold difference between the extremes). The mRNA concentrations for some strains (e.g., 5'UTR_250_4 and 5'UTR_1,000_11) were extremely low. Although high mRNA concentrations were only observed for 5'UTRs with high RBS indexes (≥ 33,000), there was no clear correlation between *lacZ* mRNA concentration and RBS index. For instance, strains in the same 5'UTR-33 k series with RBS indexes around 33,000 had mRNA concentrations that differed by a factor of 8 (Figure 2).

We next examined the relationship between protein activity and mRNA level to explore how changes in mRNA concentrations affect the activity of the protein and by approximation its concentration (Figure 3A). As expected (Guimaraes et al., 2014),

β-galactosidase activity was positively related to *lacZ* mRNA levels (R² = 0.47; Figure 3A). The moderate correlation suggests that translation and mRNA concentration regulation are both involved in the regulation of protein activity. Pairwise comparisons between the 5'UTRs revealed various scenarios. There were pairs in which the concentration of mRNA was the main determinant of protein activity. For instance, 5'UTR_66k_36 and 5'UTR_100k_41 differed by a factor of 2 in both mRNA level and protein activity (green line, Figure 3B). The relationship for other pairs, indicating no association between mRNA concentrations and protein activities, suggests in contrast that translation is the main determinant of protein activity. This was the case for 5'UTR_66k_37 and 5'UTR_100k_40 for instance (vertical red line, Figure 3B). The data for most pairs of 5'UTRs point toward a combination of translation and mRNA concentration regulation of protein activity, acting in the same or opposite directions. For 5'UTR_33k_29 and 5'UTR_100k_38 (blue line, Figure 3B), the difference in protein activities (2.5-fold) was twice the difference in mRNA concentrations (1.2-fold), indicating that translation and mRNA concentration regulation both increase protein activity. In contrast, between 5'UTR_33k_30 and 5'UTR_66k_37 (pink line, Figure 3B), the increase in the protein activity (+20%) was four times lower than the increase in the mRNA concentration (+80%), indicating that the difference in translation between these strains counteracts the positive effect of the increased mRNA concentration.

Altogether these results demonstrate that 5'UTR sequences affect gene expression both at the translational level and through mRNA concentrations. Intracellular mRNA concentrations are the balance of synthesis *via* transcription and degradation. Since all the constructs had the same level of transcription induction, we next investigated 5'UTR-mediated changes in mRNA stability.
<figure><img src="image_5.png" /><figcaption>FIGURE 2</figcaption></figure>

lacZ mRNA concentrations associated with 41 synthetic 5'UTRs ordered by RBS index. The lacZ mRNA concentration measured in each strain is reported as a fold change relative to the reference strain (5'UTR_33k_30). Error bars represent standard deviations (n=3).

<figure><img src="image_6.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>FIGURE 3</figcaption></figure>

Relationships between β-galactosidase activity and *lacZ* mRNA concentration for the 41 constructs. mRNA concentrations are expressed as fold changes relative to the reference strain (5'UTR_33k_30). (A,B) Correlation between protein and mRNA levels (the black line represents the linear regression line, R² =0.47). The colored lines in part (B) highlight examples in which differences in protein levels are mainly due to translation regulation (red), mainly due to mRNA concentration regulation (green), or due to a combination of translation and mRNA concentration regulation acting codirectionally (blue) or in opposite directions (pink). Error bars represent standard deviations (n =3).

Effect of 5'UTR sequences on mRNA stability

mRNA stability measurements were performed for 8 of the 41 synthetic 5'UTR sequences with a very high correlation between β-galactosidase expression and *lacZ* mRNA concentrations (R² = 0.91), indicating a predominance of mRNA concentration regulation (5'UTR_500_07, 5'UTR_1,500_14, 5'UTR_500_09, 5'UTR_1,500_15, 5'UTR_2,500_16, 5'UTR_33k_28, 5'UTR_33K_30

and 5'UTR_66k_35; Supplementary Figure S1A). The decrease in *lacZ* mRNA concentration over time was measured after transcription initiation was blocked by adding rifampin. The half-life of *lacZ* mRNA varied between 0.4 ± 0.1 min and 2.3 ± 0.1 min in these constructs (Figure 4), confirming that 5'UTR sequences affect the stability of the corresponding transcript.

We next analyzed whether these changes in mRNA stability depended on the nature of the downstream reporter gene. Half-life measurements were repeated (Figure 4) after
<figure><img src="image_8.png" /><figcaption>FIGURE 4</figcaption></figure>

Half-lives of mRNAs fused to the eight selected 5'UTRs and graphical interpretation of the effects of the 5'UTRs on mRNA stability and concentration. Red arrow, an increase in mRNA half-life associated with an increase in mRNA concentration; blue arrow, similar mRNA half-lives but an increase in mRNA concentration; green arrow, a longer mRNA half-life not reflected by an increase in mRNA concentration. Error bars represent standard deviations (n = 3).

replacing the downstream gene (*lacZ*) with either *txAbF* or *msfGFP*. As observed with *lacZ*, the half-lives of *txAbF* and *msfGFP* mRNAs differed between strains with different 5'UTRs. Although 5'UTR_33k_30 was associated with the highest mRNA half-lives with all three reporter genes, the ranking of the 5'UTRs and the magnitude of the variations in half-life (5-fold for *lacZ*, 6-fold for *txAbF*, and 2.5-fold for *msfGFP*) differed between reporter genes.

These results show for three different reporter genes that changing the 5'UTR sequence can affect mRNA stability. However, the magnitude of this effect depends on the nature of the downstream reporter gene.

## Effect of 5'UTR-mediated changes in mRNA stability on mRNA concentration

We then sought to determine whether 5'UTR-dependent changes in mRNA stability could explain differences in mRNA

concentrations and protein activity. We first verified for the eight selected 5'UTRs that protein activity/level and mRNA level were as highly correlated with *txAbF* and *msfGFP* as reporter genes as with *lacZ* (Supplementary Figures S1B,C), confirming that protein expression was mainly regulated by the mRNA concentration. It can be noted that both the good protein producers (5'UTR_66k_35 and 5'UTR_33k_30) and the low protein producer (5'UTR_2,500_16) contained the GGAGG SD sequence (Supplementary Table S1) indicating that the presence of a perfectly complementary SD sequence alone does not predict the protein level. We then investigated how changes in mRNA concentration were related to changes in mRNA stability. Graphical comparisons for the eight 5'UTR sequences and the three reporter genes revealed (i) variations in mRNA concentration mirroring variations in mRNA stability (red arrow, Figure 4); (ii) variations in mRNA concentration unrelated to variations in stability (blue arrow, Figure 4), which must therefore be due to differences in transcription; and (iii) similar mRNA concentrations despite variations in mRNA stability
(green arrow, Figure 4), indicating that changes in transcription must counteract the effect of increased mRNA stability.

Degradational regulation coefficients (pD) were calculated to quantify the role of mRNA stability in determining mRNA concentration (Table 1). For a given reporter gene, the contribution of mRNA stability to regulating mRNA concentration was found to vary between 5'UTR sequences. For example, *txAbF* mRNA stability strongly contributed to mRNA concentration regulation with 5'UTR_500_09 and 5'UTR_33k_30, contributed only partially with 5'UTR_500_07, 5'UTR_2,500_16 and 5'UTR_66k_35, and was not involved at all with 5'UTR_1,500_14 and 5'UTR_1,500_15. The role of mRNA stability in regulating mRNA concentration also varied with the reporter gene for a given 5'UTR. For example, changes in mRNA concentration with 5'UTR_500_09 were associated with

changes in mRNA stability for *txAbF* (strongly) and *msfGFP* (partially), but not for *lacZ*. Note that none of the 5'UTRs were associated with stability-related control of mRNA concentration for all reporters, while with 5'UTR_1,500_14 and 5'UTR_1,500_15, no regulatory effect of mRNA stability on mRNA concentration was observed for any of the reporters.

To examine whether the type of control of mRNA concentration was related to sequence features, we first analyzed the minimum free energy of a sliding window in the translation initiation region (TIR) that comprises the synthetic 5'UTR and the first 11 codons of the downstream gene (−33 to +33 bp region relative to the start codon; Figure 5). The lower the value of the ΔG, the more the formation of strong secondary structures is possible. Transcriptional control was associated with both the absence and presence of strong secondary structures in the TIR sequences (5'UTR_1,500_14 and 5'UTR_1,500_15, respectively). Degradational control appeared to be present only in the absence of strong secondary structures in the TIR sequences (local ΔG never less than −7 kcal/mol for 5'UTR_500_9 for *txAbF*, 5'UTR_66k_35 for *lacZ* and *msfGFP* and 5'UTR_33k_30 for *lacZ* and *txAbF*). The A/U richness of these TIR sequences was not significantly higher than the others to facilitate cleavage by RNase E, and the complementary SD sequence (GGAGG) and its degenerate forms were both present (Supplementary Table S1).

Altogether, these results show that for 11 of 21 constructs, the 5'UTRs contributed (strongly or partially) to the regulation of mRNA stability, which in turn regulated mRNA concentration. Five of the eight 5'UTR sequences were involved in the regulation of mRNA stability in a downstream gene dependent manner. The absence of strong local secondary structures in their TIR sequences appears to be a necessary but not sufficient element to explain the stability-related control of mRNA concentration.

<table><caption>TABLE 1 Degradational regulation coefficients (pD) of constructs with eight different 5'UTRs and three reporter genes.</caption><thead><tr><th>5'UTR</th><th>lacZ</th><th>txAbF</th><th>msfGFP</th></tr></thead><tbody><tr><td>5'UTR_500_07</td><td>0.13</td><td>0.44</td><td>0.54</td></tr><tr><td>5'UTR_1,500_14</td><td>0.13</td><td>0.37</td><td>0.23</td></tr><tr><td>5'UTR_500_09</td><td>0.37</td><td>1.08</td><td>0.56</td></tr><tr><td>5'UTR_1,500_15</td><td>0.08</td><td>0.33</td><td>0.31</td></tr><tr><td>5'UTR_2,500_16</td><td>0.03</td><td>0.48</td><td>0.46</td></tr><tr><td>5'UTR_33k_28</td><td>-</td><td>-</td><td>-</td></tr><tr><td>5'UTR_66k_35</td><td>2.6</td><td>0.44</td><td>1.39</td></tr><tr><td>5'UTR_33k_30</td><td>1.13</td><td>1.61</td><td>0.31</td></tr></tbody></table>

5'UTR_33k_28 was used as a reference. pD values are classified in three groups: high (pD > 0.6, in red), indicating that stability regulation is the primary factor explaining variations in mRNA concentration; low (pD < 0.4, in blue), indicating that stability regulation does not significantly contribute to variations in mRNA concentration; and intermediate (0.4 < pD < 0.6, in green), indicating that stability regulation and transcription regulation contribute at similar levels to variations in mRNA concentration.

<figure><img src="image_9.png" /><figcaption>FIGURE 5</figcaption></figure>

A sliding window analysis of the minimum free energy in the translation initiation region. A 33-nucleotide long sliding window was applied in the translation initiation region (TIR) of each mRNA of reporter genes fused to the eight selected 5'UTRs. The TIR sequence (−33 to +33 bp region relative to the start codon) comprises the synthetic 5'UTR and the first 11 codons of the downstream gene. The minimum free energy was calculated in each window using the mfold software. The minimum free energies of *lacZ* are represented by the purple curve, of *txAbF* by the orange curve and of *msfGFP* by the gray curve. Blue T letter: mainly a transcriptional control; Green S letter: a shared transcriptional and degradational control; Red D letter: mainly a degradational control. 5'UTR_33k_28 was used as a reference for the study of the degradational/transcriptional controls.
Discussion

This study provides evidence that 5'UTR sequences play a key
role in regulating gene expression in *E. coli*. Our results show that
5'UTRs can influence protein activity/level by regulating
translation initiation, mRNA stability and mRNA concentration.
We observed that the highest and lowest β-galactosidase protein
activities were obtained for 5'UTRs with the highest and lowest
theoretical translation initiation rates. Because β-galactosidase
protein activity is a good proxy for its concentration, these results
confirmed the key role of translation initiation in determining
protein levels in these cases. However, for many 5'UTR sequences,
the theoretical translation initiation rate was a poor predictor of
the resulting protein level. We found that *lacZ* mRNA
concentrations are regulated in a 5'UTR-dependent manner, and
as shown by the moderate correlation between mRNA and protein
levels, that this regulation mechanism in turn influences protein
levels, often in association with translational regulation.

For two of the 5'UTRs (5'UTR_66k_35 and 5'UTR_33k_30),
changes in *lacZ* mRNA concentrations mirrored strong changes
in mRNA stability. The fact that high mRNA concentrations were
only observed for 5'UTRs with high RBS indexes supports the
hypothesis that translation initiation has an indirect effect on
mRNA stability. We also showed that 5'UTR-dependent mRNA
stability was dependent on the nature of the downstream gene.
Translation initiation region (5'UTR + first 11 codons of the
downstream gene) with efficient translation initiation rates
recruit more ribosomes to the mRNA, and the high ribosome
occupancy may protect the transcript from degradation by
RNases, thereby increasing the mRNA concentration (*Iost and
Dreyfus, 1995*). Stability regulation does not appear to be related
to the strength of the SD sequence, but it does appear to require
the absence of strong local secondary structures in their TIR
sequences, an absence that may indeed facilitate translation
initiation. A downstream gene-dependent 5'UTR effect on
mRNA concentration has been reported previously (*Mutalik
et al., 2013b*), but he did not measure mRNA stability. The context
dependence of the 5'UTR effect is a great challenge for synthetic
biology because ideally, the effects of standardized regulatory
elements should be independent of context. This has motivated
the design of new 5'UTR-downstream gene junctions based on
overlapping genetic elements with translation coupling, to ensure
translation initiation is independent of the nature of downstream
genes (*Mutalik et al., 2013a*).

Interestingly, two 5'UTRs never regulated mRNA
concentration through mRNA stability for any of the reporter
genes. Although all the studied constructs were controlled by
the same promoter and had similar levels of transcriptional
induction, the absence of stability regulation indicates that
these two 5'UTRs must regulate mRNA concentration through
transcription, possibly by acting on RNA polymerase escape
from the promoter (Heyduk and Heyduk, 2018). The initially
transcribed sequence (ITS) corresponding to the first 20
nucleotides of the 5'UTR has been reported to increase or slow

down the escape of the RNA polymerase promoter and thus
participate in the initiation of transcription (Heyduk and
Heyduk, 2018). Exploring this hypothesis would require
measurements of sequence-directed RNA polymerase pausing
and RNA/DNA duplex stability in the ITS, which are beyond
the scope of this study.

In conclusion, this study demonstrates that synthetic 5'UTR
sequences can be used as "regulatory-hubs" of gene expression,
as they act at the level of translation and of mRNA degradation
and transcription. The complexity arising from overlapping
regulation mechanisms and the dependence of the 5'UTR effects
on the downstream gene make protein levels difficult to predict
for any given construct in the context of synthetic biology. Study
of more combinations of 5'UTRs and reporter genes is required
to fully understand the rules governing protein level regulation
by 5'UTR sequences in E. coli.

Data availability statement

The original contributions presented in the study are included
in the article/Supplementary material, further inquiries can
be directed to the corresponding authors.

Author contributions

FC performed the experiments and wrote the first draft of the
manuscript. SN, LG, and MC-B contributed to conception and
design of the study and wrote the manuscript. All authors
contributed to manuscript revision, read and approved the
submitted version.

Funding

This work was supported by the French National Research
Agency [ANR-18-CE43-0010] and Award of a Doctoral
Fellowship (FC) from the China Scholarship Council.

Conflict of interest

The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.

Supplementary material

The Supplementary material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2022.1088941/full#supplementary-material

SUPPLEMENTARY FIGURE S1

Correlations between protein and mRNA levels of reporter
genes fused to the eight selected 5'UTRs (A) for lacZ,
R² = 0.91; (B) for txAbF, R² = 0.93; and (C) for msfGFP,
R² = 0.87 The eight selected constructs were 5'UTR_500_07,
5'UTR_1_500_14, 5'UTR_500_09, 5'UTR_1_500_15,
5'UTR_2_500_16, 5'UTR_33k_28, 5'UTR_33k_30 and
5'UTR_66k_35. The black lines are linear regression fits.
Error bars represent standard deviations (n = 9 biological
triplicates and technical triplicates for measurements of
β-galactosidase and α-L-arabinofuranosidase activity and msfGFP
fluorescence/OD, and n = 3 for measurements of
mRNA concentration).

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