Signal Recognition Particle: the RNA key to protein secretion

Signal Recognition Particle: the RNA key to protein secretion 

Proteins are found everywhere within a cell. Many localize in the cell membranes, where they play crucial roles in everything from signal reception and transduction to ion balance and ATP synthesis. But how do new proteins get incorporated into the membranes from their origin of translation in the cytosol? One of the ways that the cell does this is through the use of co-translational systems that enable a protein to be translated by a ribosome while the unfolded polypeptide chain is thread through a channel protein in a membrane. There are a variety of systems that perform this function, but the most conserved system found in prokaryotes and eukaryotes is SRP-RNC-Sec61/SecYEG.

The SRP-RNC-Sec61/SecYEG pathway involves the use of signal recognition particle (SRP), signal receptor (SR), ribosome-nascent chain complex (RNC), and the Sec61 (or SecYEG in eukaryotes) membrane channel protein. The RNC refers to a ribosome attached to a polypeptide chain that it is translating.  The process begins when the polypeptide end of an actively translating RNC is recognized by SRP (Akopian et al., 2013). SRP binds to the RNC and causes a pause in translation. When SRP associates with SR at a cell membrane, it delivers the ribosome and the unfolded polypeptide chain to the Sec61 channel protein in the target membrane. Channel proteins span lipid bilayers and passively transport specific molecules through the membrane when signaled to open. When the RNC binds to Sec61, it resumes translation and either integrates the polypeptide chain into the lipid bilayer or funnels it across the membrane to enter the secretory pathway. The SRP and SR then disassociate the restart the cycle (Akopian et al., 2013). While this system is highly conserved in both prokaryotes and eukaryotes, there are subtle differences. For simplicity, this blog post will focus on the prokaryotic SRP-RNC-Sec61 pathway.

 Figure 1: Different methods of cell protein secretion. This blog post is focusing on the central pathway that involves SRP binging RNC and pausing translation. The SRP-RNC complex then associates with SR and is brought to the SecYEG channel membrane protein (Akopian et al., 2013). 

In bacteria, SRP is composed of a protein called Ffh that is bound to an SRP RNA. The Ffh protein has two regions that are important for co-translation: the M domain and the NG domain.  The M domain is responsible for recognizing and binding to the SRP RNA. The NG domain of Ffh is a GTPase that interacts with the homologous SR NG domain to form a heterodimer. While Ffh is important for the binding the SRP RNA and binding to the SR protein, the SRP RNA is what allows SRP to function.

The SRP RNA is a double-stranded 4.5S RNA that mediates global reorganization of the SRP in response to RNC and SR binding, allowing for rapid RNC-SRP-SR complex assembly at Sec61.  Before it binds to the RNC, SRP can take on in a variety of conformations due to a flexible linker that spans two domains of the Ffh protein. However, after binding to RNC, the SRP RNA is oriented so it lies parallel to the surface of the ribosome (Akopian et al., 2013). This reorientation is thought to expose a part of the SRP RNA involved in interactions with SR. Additionally, when the SRP-RNC complex binds to SR, the NG domains on both Ffh and SR are reconfigured and shifted towards the distal end of the SRP RNA. This reorientation exposes the ribosome exit site, making it easier for the RNC to bind with Sec61 and efficiently deliver its unfolded polypeptide cargo (Akopian et al., 2013). The SRP RNA is essential for SRP function by directing a series of structural rearrangements that allows the other components of the pathway to bind to the RNC-SRP complex.

Figure 2: Cartoon structures of bacterial SRP and SR. SRP is made up of a Ffh protein (M and NG domains) and a 4.5S RNA. The M domain binds to the RNC signal sequence with the NG domain is involved in interactions with SR (Akopian et al., 2013). 

Figure 3: SRP RNA reorganization during the SRP-SecYEG pathway. Unbound SRP does not have a universal tertiary structure (B) until it binds to RNC, where it undergoes a conformational change to attach to the ribosome (C). When SRP-RNC binds to SR (D), the SRP-SR NG domain relocalizes to the distal end of the RNA to allow for unloading RNC cargo (Akopian et al., 2013).

Lately, researchers have been investigating SRP as a novel antibiotic target. Antibiotic resistance is a global epidemic that has been an important topic of research. Often, antibiotic targets focus on bacterial cell walls or key biosynthesis enzymes. While these are initially successful, the lack of diversity in antibiotic targets means that when a bacteria becomes resistant to one antibiotic, the mutation often confers resistance to other antibiotics as well (Faoro et al., 2018). Therefore, novel antibiotic targets, such as SRP, are of high interest to the scientific community that is working to fight this epidemic. Studies have shown that despite structural differences between prokaryotic and eukaryotic SRP, prokaryotic SRP can replace eukaryotic SRP in vivo and maintain efficient protein targeting (Akopian et al., 2013). Due to its conserved nature and biological importance, SRP is a strong and suitable antibiotic target. Additionally, mutations or deletions in parts of SRP responsible for RNC and SR interactions that stopped endogenous activity resulted in cell death or severe functional impairment (Faoro et al., 2018). By using fragment-based drug discovery (FBDD), a method that involves identifying small chemical fragments with initially weak millimolar affinities to the target site and then developing them to have a stronger affinity, Faoro et al. (2018) found at least one fragment of SRP that showed antimicrobial activity. This result is highly promising for the development of SRP based antibiotics.

Figure 4: Antibiotic growth inhibition assays. SRP fragments were spotted on filter paper and incubated overnight on E.coli cultures with Kanamycin serving as a positive control. Fragment 2 showed greater inhibition than fragment 1 or 3, but it was significantly less than the control. The observation of some antibiotic inhibition is promising although potency needs to be optimized (Faoro et al., 2018).
   
The SRP-RNC pathway is essential for protein localization and secretion within cells. It is found in both prokaryotes and eukaryotes and shares many components that have been preserved by evolution, such as the SRP RNA. Because of this, the SRP is a novel antibiotic target that offers a potential solution to the epidemic of antibiotic resistance facing the world today.

Meet the Author

Sé is a junior at Mount Holyoke who is graduating in 2019 with a bachelor’s degree in Biology and a minor in Chemistry. She currently works in Professor Amy Camp’s lab at Mount Holyoke College, where she is studying bacterial protein interactions. Sé plans to continue with research in the biological field when she graduates.

Literature cited:

Akopian, D., Shen, K., Zhang, X., Shan, S. (2013). Signal Recognition Particle: An
             essential protein targeting machine. Annual review of biochemistry. 82, 693-721.
Faoro, C., Wilkinson-White, L., Kwan, A. H., Ataid, S. F. (2018). Discovery of fragments that target              key interactions in the signal recognition particle (SRP) as potential leads for a new class of                antibiotics. PLOS: One.
Halic, M., Becker, T., Pool, M. R., Spahn, C., Grassucci, R., Frank, J., Beckmann, R.
             (2004). Structure of the signal recognition particle interacting with the elondation-arrested                  ribosome. Nature. 427, 808-814.