Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology

Abstract

Gene expression occurs in two essential steps: transcription and translation. In bacteria, the two processes are tightly coupled in time and space, and highly regulated. Tight regulation of gene expression is crucial. It limits wasteful consumption of resources and energy, prevents accumulation of potentially growth inhibiting reaction intermediates, and sustains the fitness and potential virulence of the organism in a fluctuating, competitive and frequently stressful environment. Since the onset of studies on regulation of enzyme synthesis, numerous distinct regulatory mechanisms modulating transcription and/or translation have been discovered. Mostly, various regulatory mechanisms operating at different levels in the flow of genetic information are used in combination to control and modulate the expression of a single gene or operon. Here, we provide an extensive overview of the very diverse and versatile bacterial gene regulatory mechanisms with major emphasis on their combined occurrence, intricate intertwinement and versatility. Furthermore, we discuss the potential of well-characterized basal expression and regulatory elements in synthetic biology applications, where they may ensure orthogonal, predictable and tunable expression of (heterologous) target genes and pathways, aiming at a minimal burden for the host.

Keywords: RNA polymerase; attenuation control; regulatory RNA; sigma factors; synthetic biology; transcription factors.

Figure 1.

Bacterial transcription and translation is coupled. (A) Simplified schematic view of mRNA and protein synthesis with several RNA polymerase (RNAP) molecules simultaneously transcribing a single gene, and several ribosomes translating a single monocistronic mRNA. Transcription starts with binding of RNAP holoenzyme to the promoter region, mRNA synthesis proceeds in the direction 5΄ to 3΄ and stops at a Rho-dependent or -independent terminator. In both instances, transcription termination is accompanied by release of the mRNA molecule and dissociation of RNAP from the DNA template. Notice that during transcription elongation σ (blue colored) may (as in the figure) or may not dissociate from the complex. Translation by fully assembled 70S ribosomes (after initial binding of the 30S subunit to the RBS) starts at the initiation codon (mostly AUG, positioned in the P site of the ribosome), proceeds in the direction N-terminus to C-terminus and stops at a nonsense codon. Translation termination is accompanied by the release of the polypeptide chain from the tRNA and recycling of the ribosomes in separate 30S and 50S subunits. The magenta colored segment near the 5΄-end of the mRNA represents the ribosome-binding site (RBS) that comprises the Shine–Dalgarno sequence and the initiation codon. 5΄-UTR and 3΄-UTR correspond to untranslated regions of the mRNA near its 5΄- and 3΄-end, respectively. For simplicity, initiation, elongation and termination factors are not shown (see Box 1 for more information on bacterial transcription and translation). (B) Schematic representation of the RNAP holoenzyme composed of the core enzyme (α₂ββ΄ω) and a σ factor that is responsible for promoter recognition. UP is the upstream promoter element that is contacted by α-CTD, whereas the –10 and –35 promoter elements are contacted by different parts of σ (see Fig. 2C for details). Ext represents the extended –10 promoter element and Disc the discriminator site. TSS represents the transcription start, which is mostly a purine. Sequences and distances correspond to the consensus promoter for the housekeeping σ factor (σ⁷⁰) of E. coli. (C) Schematic view of an elongating fully assembled 70S ribosome with three binding sites for tRNA molecules. A, the aminoacyl site with an incoming aminoacylated tRNA selected on basis of codon–anticodon complementarity; P, the peptidyl site with a tRNA carrying the growing peptide chain; and E, the exit site for binding of an uncharged tRNA after transfer of the growing peptide chain from the P site to the A site-bound tRNA followed by a translocation cycle.

Figure 2.

Domain composition of σ factors and their interaction with cognate promoter sequences. (A) The seven σ factors of E. coli, which all bind competitively to similar regions of the unique core RNAP but interact with specific promoter sequences centered around positions –10 and –35 for the six members of the σ⁷⁰ family and around –12 and –35 for σ^N, the sole representative of the σ⁵⁴ family. Notice that some cross-talk may exist in the binding of alternative sigma factors to non-cognate promoter sequences as observed for the housekeeping σ⁷⁰ and σ^S in E. coli and among members of the ECF (extracytoplasmic function) group of σ factors in general. (B) Function and division in four groups of the six σ⁷⁰ family members of E. coli based on domain composition, and σ⁵⁴ that forms a distinct family on its own. (C) Promoter structure and domain composition of the four groups of σ⁷⁰ family members. NCR stands for non-conserved region. Arrows indicate interactions of specific subdomains of σ with promoter sequences. σ_1.1 plays an inhibitory role in promoter binding. (D) Promoter structure and domain composition of σ⁵⁴. RpoN is the region that specifically interacts with the –24 promoter region and is the most conserved domain among σ⁵⁴ proteins. HTH stands for the helix-turn-helix motif that interacts with the –12 promoter sequence, and ELH for extra long helix. CBD is the RNAP core-binding domain. RI interacts with RIII and plays an inhibitory role, blocking the entry of the DNA template strand. It is also a site of contact for activator proteins. RII penetrates deeply in the DNA-binding channel and also plays an inhibitory role and has to be displaced in the transcribing complex.

Figure 3.

Various mechanisms for regulation of σ factor activity. (A) Schematic representation of four mechanisms for regulation of σ-anti-σ factor activity involving stress-induced release of the σ factor from the inhibitory σ-anti-σ complex: regulated intramembrane proteolysis (RIP) of the anti-σ factor, partner switching, direct sensing and reduction of anti-σ concentration by secretion through the flagellar export system. Red colored symbols represent proteases. Orange colored symbols represent σ factors belonging to different groups (group 3 represented with three domains and group 4 (ECF) represented with two domains). A black colored symbol represents the cognate anti-σ factor. A blue colored ellipse with a P represents a phosphorylated anti-anti-σ factor. Zn surrounded by a green colored sphere represents a zinc atom. (B) Scheme summarizing different mechanisms used for regulation of σ factor activity in various bacteria. (C) Synthetic orthogonal gene expression system for E. coli based on the introduction of heterologous or artificial (hybrid) σ factors and their cognate specific promoters exhibiting no cross-talk, and fine-tuning of gene expression by use of promoter libraries with a broad range of transcription initiation frequencies without loss of orthogonality.

Figure Box 2.

Various types of logic gates. A and B represent inputs (with absence of input indicated with 0 and presence with 1), and X the potential output.

Figure 4.

Regulation of promoter activity by DNA-binding transcription factors. (A) Negative regulation by repressors (red colored symbols). Various possibilities are depicted. In repression by steric hindrance, binding of the repressor in overlap with the RNAP-binding site generates a direct competition in the binding between the regulator and the RNAP. In the roadblock model, binding of the repressor downstream of the promoter element physically inhibits the further progression of the RNAP. Pronounced DNA deformation may result from the binding of repressor proteins upstream and downstream of the promoter making the latter unsuitable for RNAP binding. Negative regulation by anti-activation may result from the mutually exclusive binding of a repressor and an activator to overlapping sites (not shown) or, as shown, from the interference of the repressor with the stimulating effect of the activator on RNAP recruitment. Finally, promoter clearance may be inhibited by a repressor protein that makes strong contacts with both DNA and RNAP thus inhibiting RNAP movement. (B) Stimulation of transcription initiation by activator proteins (green colored symbols). In class I activation, the activator binds at variable distances upstream of the –35 promoter element and makes protein–protein contacts with one or two σ-CTDs, thereby stimulating RNAP recruitment. In class II activation, the regulator binds in overlap with the promoter and makes contacts with α-NTD or σ, or with both. Some activators bind in between the –10 and –35 sequences of promoters with a suboptimal spacing and stimulate promoter activity by untwisting the DNA helix, thus generating a better alignment of the promoter elements for RNAP binding. σ⁵⁴-dependent promoters require activators of the AAA⁺ family and the energy of ATP hydrolysis. They generally bind upstream of the promoter elements and act in conjunction with a DNA-bending protein (purple sphere) to facilitate their contact with the RNAP.

Figure 5.

Theoretical examples of synthetic circuit building for orthogonal gene expression. (A) Hypothetical example of a three-level circuit for orthogonal gene expression combining various gates and integrating multiple signals. The modules on the left represent two AND gates in which inputs I₁ and I₂ allow the synthesis of a TF and its chaperone (green and salmon colored symbols) that will activate the production of the output O₁ (magenta colored), whereas inputs I₃ and I₄ ensure the synthesis of the two parts of a split TF (or alternatively of a split single subunit RNAP) (purple and light blue colored symbols), which will result in the production of output O₂ (taupe colored). O₁ and O₂ form a hetero-oligomeric TF that will allow the production O₃, an orthogonal alternative σ factor (orange colored) that eventually will ensure the synthesis of the final outcome O₄. However, this will only occur in the absence of the inputs I₅ and I₆. In the presence of I₅, the formation of the hetero-oligomeric activator (O₁-O₂) will be inhibited by sequestration of O₂ upon binding with an alternative binding partner (dark blue colored), whereas in the presence of I₆ the orthogonal σ factor will be sequestered by its cognate anti-σ factor (black colored). (B) Orthogonal gene expression based on toehold switches with co-localized RNA sensing and output modules. In toehold switches, the RBS is not accessible for ribosome binding, unless the RNA secondary structure is disrupted by interaction of the mRNA with a complementary synthetic sRNA. Importantly, and in contrast to other regulatory mechanisms operating at translational level (see Figs 6–8), in toehold switches the RBS is not part of the double-stranded RNA structure, which allows more flexibility in the design of the switch and its cognate synthetic sRNA (Green et al.2014). In the example shown here, synthesis of one or more synthetic sRNA in the cell will allow ribosome binding and translation of the cognate ORFs (here represented by red, yellow, green and blue fluorescent proteins).

Figure 6.

Regulation of mRNA stability and regulation of bacterial translation and transcription with RNA-binding proteins (RBP). (A) Regulated access to ribonucleases. A RBP (blue colored) may directly compete with the binding of a nuclease (yellow colored) to an overlapping site on the mRNA, liberate a recognition site for a ribonuclease on the mRNA resulting in a negative effect on gene expression or favor the formation of a secondary structure in which the target site (yellow colored box) for the ribonuclease is not readily accessible. The latter results in a positive effect on gene expression. (B) Regulated translation initiation. A RBP may (i) directly compete with the ribosome (30S subunit) for binding to the RBS (magenta box), (ii) favor the formation of structure in which the RBS is trapped in a double-stranded secondary structure, (iii) favor the formation of a secondary structure that liberates the RBS, hence facilitating ribosome binding and stimulating translation, (iv) inhibit translation initiation by stabilizing a complex in which the 30S subunit of the ribosome is trapped in an incompetent state by the RNA and (v) indirectly inhibit translation initiation by generating a steric clash with ribosome binding to the RBS. Green colored rectangles represent ORFs. (C) Improved access to proteases by the chaperone function of the RBP that may act in conjunction with a small regulatory RNA (green line) complementary to the mRNA. (D) Transcriptional attenuation. A RBP may positively or negatively affect premature transcription termination by stabilizing a terminator structure (dark red colored hairpin), or, inversely, by stabilizing an anti-terminator structure (sea-green colored hairpin), or still by exposing a recognition site for a transcription terminator protein such as Rho (bright red colored) (or alternatively a target site for a ribonuclease).

Figure 7.

Riboswitches: RNA sensors and RNA thermometers. (A) Ligand (magenta sphere) induced formation of transcriptional terminator or anti-terminator RNA structures in the mRNA (green colored) that result in premature transcription termination of the RNAP (gray colored) or allow downstream transcription elongation, respectively. (B) Translational attenuation mechanisms whereby ligand binding traps the RBS (magenta box) in a RNA secondary structure and is not accessible for ribosome (yellow colored) binding or, inversely, stabilizes an alternative structure and frees the RBS, thus allowing initiation of translation. (C) RNA thermometer. Here the RBS is trapped in a secondary structure and not available for ribosome binding. Melting of this structure in a zipper-like manner at increasing temperatures liberates the RBS, thus allowing translation initiation. (D) Reversible and temperature-dependent formation of alternative secondary RNA structures that may affect premature transcription termination or translation initiation.

Figure 8.

Regulation by small RNAs (sRNAs). (A) Regulation of mRNA translation (top part) and transcription attenuation (bottom part) by cis-acting antisense sRNAs. A cis-acting sRNA (blue colored) encoded from the opposite strand as its target gene (green colored) inhibits gene expression at the post-transcriptional level by sequestering the ribosome-binding site (magenta colored rectangle) in a double-stranded RNA secondary structure. Transcriptional attenuation results from sRNA induced formation of a terminator (attenuator; red colored hairpin) structure in the mRNA at the expense of the anti-terminator. (B) Three methods illustrating gene regulation by trans-acting sRNAs. Trans-acting sRNAs are only partially complementary to the target RNA and frequently require the help of a chaperone protein (blue ellipse) such as Hfq in E. coli for stabilization, folding and target binding. Due to the limited bp complementarity with the target, trans-acting sRNAs may bind several distinct mRNAs and either inhibit translation by sequestering the RBS, which may also lead to accelerated mRNA degradation (top part), or inversely free the RBS and hence stimulate translation (middle part). Finally, trans-acting sRNAs bearing multiple binding sites for an inhibitory RNA-binding protein (RBP, blue colored ellipse) indirectly stimulate translation of one or more mRNAs by titration/sequestration of the RBP (bottom part).