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Degrons

Anna Stikāne

What are degrons?

Synthetic biology aims to build rigid, orthogonal molecular systems with valuable industrial and medical applications. This implies ability to regulate protein production (transcription and translation of mRNA) as well as their lifespan. A convenient method of controlling protein lifetime is marking them for protease-mediated degradation. In bacteria this can be achieved utilizing a widely studied and conserved SsrA-tag mediated protein degradation system. Natural SsrA-tag is added to nascent polypeptides as a way to rescue stalled ribosomes (a process termed trans-translation).¹ SsrA tagged proteins are recognised and degraded by proteases within all cellular compartment (i.e., ClpXP and ClpAP in cytoplasm, FtsH within membranes and Tsp in periplasmic space).^2–4 The actual degradation rate of SsrA tagged proteins depend on several factors including the sequence of SsrA polypeptide, SsrA binding protein concentration, competition with other interacting molecules and temperature, which all might be altered and engineered to get even more robust and predictable degradation of SsrA tagged proteins.^5–9 Engineered protein instability can be advantageous to get specific protein concentrations in vivo and prevent unwanted protein accumulation. This in turn is beneficial for synthetic genetic constructs were protein production timing and concentration thresholds are crucial, e.g., genetic bi-stable switches, oscillators, biosensors, and faster acting bacterial logic gates and computation systems.

SsrA-tags and protein degradation

The natural function of SsrA

In Escherichia coli SsrA (EcoCyc¹⁰ accession number EG30100) is 362 nucleotides long monocistronic gene, encoding for small stable 10Sa RNA.¹¹ SsrA RNA is also referred to as transfer-messenger RNA or tmRNA as it has both mRNA and tRNA structural properties.¹² The biological function of SsrA RNA is to recognise and resolve ‘troubled’ translation via process called trans-translation.¹ The molecular model of trans-translation was proposed by Keiler et al. in 1996¹ and since then has been further explored and reviewed in much greater details.^13,14 To summarize, tmRNA as tRNA is charged with alanine and binds to stalled mRNA-ribosome complexes. Then ribosome switches templates from mRNA to incoming SsrA and finishes the translation.(Figure 1) This process creates a fusion protein ending with 11 amino acids long ‘AANDENYALAA’ peptide tag.^1,15 The added SsrA tag then is recognised and rapidly degraded by ClpXP and ClpAP cytoplasmic proteases, FtsH(HflB) membrane protease and Tsp (Prc) periplasmic protease thus preventing build-up of incorrect proteins in all cellular compartments.^2–4

Cytoplasmic degradation of SsrA tagged proteins

The SsrA-tagged protein degradation can be subdivided into 5 steps: (1) substrate binding to ClpXP or ClpAP, (2) substrate denaturation, (3) translocation; (4) proteolysis, (5)release of peptide fragments.¹⁶ (Figure 2)

In cytoplasm SsrA tagged proteins are degraded by ClpXP and ClpAP.8 ClpX and ClpA are heptameric unfoldases that can both bind directly to SsrA tag.17 In addition, SsrA-tagged proteins can also be directed to ClpXP by ClpX adaptor protein SspB, which physically tethers SsrA-tagged substrates to ClpX.¹⁸ (Figure 3)

Then ClpX and ClpA use ATP energy to unfold the tagged protein17. ClpXP uses ~150 molecules of ATP to denature one molecule of substrate however in the absence of ClpP protease even more ATPs are hydrolysed.¹⁹ Interestingly, ClpXP degrades all SsrA-tagged substrates with remarkably similar rates although hyperstable substrates do slow down the ClpXP ATP hydrolysis rate.¹⁹

The denatured protein is then degraded by a serine peptidase ClpP.¹⁷ ClpP consists of proteolytic cavity enclosed by two stacked heptameric rings.¹⁹ The translocation of substrate into ClpP cavity requires association with ATPases like ClpX and ClpA.¹⁹ In addition, proteins unfolded by ClpX and ClpA have higher affinity for degradation by other cellular proteases.⁸ The analysis of the ClpXP activity dynamics has identified that substrate denaturation is the slow rate limiting step and the actual proteolysis step is very fast (small peptides are degraded with rate >104 min-1).¹⁶(Figure 2) For more information on ClpXP action see review from Baker and Sauer, (2012).²⁰

Engineering SsrA based protein degradation system

SsrA-tags are powerful system for controlling protein lifetime. As the tag consists of only 11 amino acids, the tag-encoding nucleotide sequence can be easily added to any protein coding sequence in a single PCR reaction. The protein structural stability or function is not affected by the attached SsrA-tag.²² Moreover, the SsrA tag recognition and following substrate denaturation and degradation by ClpXP and ClpAP is not significantly affected by the substrates structural features and stability.¹⁹ In theory this should mean that the minimal SsrA-tagged protein turnover rate within cell will be the SsrA mediated degradation rate. The actual degradation rate for SsrA tagged protein still might be higher if the protein normally is unstable. Thus SsrA-tags could be used to get better control over protein lifetime, which might be especially useful when working with such hyperstable proteins as GFP. In fact several parameters of the system can be further explored and modified to gain a broader spectrum of specified degradation rates. These include altered sequences of SsrA tag and regulation of SspB, ClpXP and ClpAP concentrations.

Yet another useful feature of the SsrA tag based system is the conservation and presence of SsrA tmRNA throughout the bacterial kingdom including plastid and mitochondrial genomes of some eukaryotes (e.g., algae and protists, respectively) which is no surprise given the importance of SsrA tag biological function as ‘translation quality checkpoint’.¹³ Table 1 from Karzai et al.,(2000) summarizes phylogenetic information of SsrA tmRNA and associated proteins within eubacterial genomes.²¹ This in turn means that an optimal SsrA tag motif can be found and used for each particular bacterial chassis.

Protein interaction information within SsrA tag

Extensive amino acid mutagenesis studies have revealed that protease recognition elements are densely packed within SsrA peptide.¹⁷ (Figure 4) Periplasmic Tsp and membrane FtsH both recognise final hydrophobic amino acids at the C-terminus.^3,4 Similarly, cytoplasmic ClpX also recognise the final three amino acids (9-11) as well as the carboxyl group.5,17 In contrast, ClpX enhancer protein SspB recognises first few amino acids (1-4) and amino acid number 7(Tyr).¹⁷ In particular, the third amino acid (N; Asn3) is crucial for SspB binding and can only be substituted with H(His) without losing SspB binding. ^17,22 The other cytoplasmic protease ClpA interacts with amino acids 8-10 and 1-2 at the degron N terminus.¹⁷ Knowledge of these protein recognition determinants can be exploited to create synthetic SsrA tags with modified amino acid composition.

Modifying the SsrA tag

Andersen et al., (1998) showed that changing last three amino acids of the tag can significantly modify the rate of degradation and are often used to specify particular version of the tag.⁵ Andersen et al., (1998) tested GFP with five different versions of SsrA-tag and reported the wild type SsrA-LAA and SsrA-LVA leading to the fastest degradation, and SsrA-AAV and SsrA-ASV resulted in slower degradation in E. coli.⁵ Same GFP-SsrA tagged constructs were tested in Pseudomonas putida and showed that SsrA-LVA provided the highest degradation rate.5 Amino acid alterations in the rest of SsrA tag can also be used to change SsrA recognition by SspB and ClpA. As SspB causes ClpX to bind more tightly to SsrA tag, SspB improves efficiency of degradation at low substrate concentrations.²³ However, SspB and ClpX recognition determinants are located very close within the tag and can lead to steric clash and impaired degradation if both proteins try to bind to the tag.6 Hersch et al., (2004) showed that extending the SsrA tag by repeating amino acids 6-7 (NY) reduced the steric clash between SspB and ClpX and created synthetic degron ‘AANDENYNYALAA’ with 4-5 times higher degradation rate than the natural SsrA LAA without changing K_M and V_max constants for ClpXP.⁶ (Figure 5) Our tested SsrA-DAS+2 tag contains two extra amino acids preventing supposed clash between SspB and ClpX and together with SsrA-DAS tag are not expected to be recognised by ClpX.⁷

Degradation rate and protease concentrations

Another factor affecting SsrA degradation rate is the concentration of intracellular proteases. Farrel et al., (2005) monitored degradation rate of SsrA tagged GFP in vivo and reported increase in degradation (rate) upon cell entry in stationary growth phase.⁸ This coincided with an increase in production and concentration of ClpA and ClpP.⁸ The concentrations of ClpX and SspB remained the same over time.⁸ One strategy to gain better control over protease abundance caused fluctuations of degradation rate is removal of protease recognition elements of SsrA. For example, McGinness et al., (2006) demonstrated a SsrA-DAS degron with a degradation rate depending only on interaction with SspB.⁷ In addition, the proposed SsrA-DAS degron system can be relatively easily transferred to other organisms because it only requires three housekeeping genes (SspB, ClpX, ClpP) and the tagged protein.⁷ Another way to control protease concentration would be increasing their production by introducing additional copy of ClpX, ClpA or SspB.

The degradation rate of SsrA tagged substrates is also influenced by molecular interactions between SsrA tag binding proteins (ClpA, ClpX, SspB) and their other substrates. For example, SspB and ClpA have overlapping recognition elements within SsrA tag thus in theory SspB could prevent ClpA binding and direct SsrA tagged proteins to degradation by ClpX.¹⁷ This has been shown in vitro but not in vivo as the amount of SspB molecules within the cell rarely outweighs ClpA.⁸ Also, molecular competition between SsrA-tag and other ClpXP and ClpAP substrates for binding ClpX and ClpA can interfere with SsrA tagged protein degradation rate. For example, another ClpA mediator ClpS can inhibit ClpAP from recognising SsrA tag.⁸

A few more factors to consider

One additional factor to consider when engineering and adding the SsrA tag is the intracellular wash out of native SsrA tags added by trans-translation. For instance, highly expressed protein with synthetic SsrA tag might occupy the bulk of ClpXP and ClpAP proteases thus impeding the removal of such natural protease substrates as damaged proteins.⁵ Such situation then in turn might lead to reduced growth rate of bacterium.⁵

The SsrA-tag mediated protein degradation rate highly depends on the temperature of environment as this system has none or minimal temperature compensation mechanisms.⁹ For example, Purcell et al., (2012) determined degradation rate of LacI with attached SsrA LVA and SsrA-LAA at 25oC and 37oC and discovered that at 37 oC LacI was degraded 3-5 times faster.⁹ To characterise SsrA degron mediated degradation dependence of temperature we performed a set of degradation rate determination experiments in four different temperatures (28⁰C, 32⁰C, 37⁰C and 42⁰C).

Future applications, usefulness

Looking from synthetic biology perspective SsrA tag system is a tool for controlling the protein lifetime. Protein instability can be advantageous for several reasons.

First of all, protein instability determines the maximum level of intracellular protein accumulation. Therefore, the amount of required proteins for optimized performance of genetic circuit (e.g., transcription factors, repressors, enzymes) can be produced by selecting the best combination of promoter, ribosomal binding site and degradation tag (e.g., SsrA tag). In addition, the protein overload within a cell can be reduced, which could be used to increase the overall stability of the genetic construct.

Secondly, controlled protein instability can be used to gain better temporal resolution of intracellular events. Fluorescent proteins (e.g., GFP, RFP, mCherry, YFP) are common and convenient reporters as they require only presence of oxygen for correct maturation and can be fused to any protein of interest without significant changes in protein properties.5 The only drawback of fluorescent proteins is their hyperstability, for example, the degradation rate for GFPmut3 has been estimated to be longer than 24 hours and the unfolding time around 20 years.^5,16 Andersen et al., (1998) attached 4 different SsrA tags and gained GFPmut3 variants with half-life ranging between 40 minutes and 2 hours.5 Thus SsrA tagged fluorescent proteins can be used to monitor dynamics of gene expression.⁵ Similarly, engineered protein instability can help to build (and in some cases have already been used) synthetic constructs where timing of intracellular events is important (e.g., synthetic oscillators24, biosensors, bacterial computing and logic gates). [Sam, hopefully we can add something of our project here as well,] However, for all these applications an optimal trade-off between maximum protein concentration (e.g., steady state fluorescence intensity) and temporal resolution must be achieved (See our characterisation results).

Considering all these advantages it is no surprise SsrA tag systems have been used in several iGEM projects generating new BioBricks (some examples summarized in Table 2) and in building new improved synthetic protein degradation regulation systems. One such example is split SspB system presented by Baker’s lab (Figure 6) and subsequently used for inspiration by iGEM13 team Bonn.²⁵

Concluding remarks

For more information on SsrA tagging and degradation system see Dr. Tania Baker Lab, which specialises and studies Clp/Hsp100 ATPases.

What did we do with them?

Measurement method

Protocols

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