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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.

What did we do with them?

Measurement method

Protocols