Molecular Mechanism of Protein Synthesis

I’ve been meaning to write this article for over a year, and now that I have a bit more time on hand, I can. While I’ve understood the cellular mechanisms for protein synthesis, as a whole; I have never been clear on the details – now I am. In this article, we will examine the cellular mechanisms that lead to protein synthesis – buckle up, the Magic School Bus is miniaturizing.

Cellular Mechanisms for Protein Synthesis

Stimulus

While this article will not focus on the number of different stimuli or “activators” of protein synthesis, I find it informative to list a few potential stimuli. In exercise, a common stimuli is mechanical and chemical through muscle remodeling and chemical stimuli leading to increased functional protein growth and density [1]. However, beyond these often-mentioned stimuli, insulin also plays a role in activating protein synthesis, as well as other cytokines (Tumor Necrosis Factor-a), growth factors (Interleukin-6) and amino acids themselves [2][3].

mTOR Signalling

The introduction of extracellular stimuli (mentioned above), leads to a series of changes within the cell that eventually, downstream, activate an enzyme named mammalian Target of Rapamycin (mTOR) [2]. mTOR is found as part of a larger protein complex (mTOR + Raptor protein) by the name mTORC1, which acts as the dominant protein synthesis activating site, but a second complex by the name of mTORC2 also contributes in a different extent [2][4].

Now, because mTOR is involved in a series of different key cellular functions, the mechanisms that interact with the enzyme can be overwhelming; however, since we are focused on protein synthesis, we can eliminate many of the other contributors. That said, we will break each of the specific mechanisms later in the article. For now, we need to know that stimuli from outside the cell either attach to the cell (insulin, TNF-a, etc.), enter the cell (amino acids, glucose, etc.), or disrupt the cell (muscular trauma) [3]. Once this occurs, there is a subsequent cascade of intercellular signals.

Once activated, however, mTORC1 phosphorylates a eukaryotic initiation factor 4E binding protein (4E-BP1), which typically, itself, phosphorylates another protein, eukaryotic initiation factor 4E (eIF4E). This phosphorylation of eIF4E, by 4E-BP1, would impede the cap-dependent translation of mRNA (essentially, it would inhibit one of the beginning steps of protein synthesis) [3][5]. However, due to mTORC1 binding to 4E-BP1, the phosphorylation of 4E-BP1 of eIF4E does not occur, releasing the “brake” on this initial step of protein synthesis [3][5]. Also, mTORC1 stimulates p70 ribosomal s6 kinases (S6K1/2) protein by phosphorylation, which increases ribosomal biogenesis and mRNA biogenesis [3].

mTOR phosphorylates Binding Protein-1 (BP1) and inactivates it from binding to Eukaryotic Intiation Factor (elF), allowing elF to further protein synthesis - explained next. 

Nuclear DNA, mRNA, & Ribosomes – Protein Synthesis Realized

First, we need to understand that DNA is the key to offering the cell the information necessary to determine which exact protein to create. This process is also largely dependent on the type of cell we are discussing – a neuron will have the DNA information, and therefore the capacity, to synthesize neurotransmitter molecules while a myocyte (muscle cell) will have the capacity to create muscle contractile proteins, and so on [6]. How does the creation of proteins for functional components of the cell occur, from DNA, however?

Initially, DNA strands, located in the nucleus of the cell, undergo a process called transcription, in which RNA polymerases (enzymes) create single stranded messenger RNA molecules (mRNA) [6]. The differentiation between different protein creations is made possible due to the fact that there are thousands of different mRNA types – with several thousand dedicated to synthesis of structural proteins and single mRNA molecules for less abundant signaling proteins [6]. mRNA then leaves the nucleus and finds its way to ribosomes on the endoplasmic reticulum (and free ribosomes) [6].

Ribosomes, interestingly, are also made up of particular RNA strands called ribosomal RNA (creative, right?) and structural proteins [6]. Ribosomes come in two parts – the top section (P60), which makes up 2/3 of the structure, and the bottom section (P40), which makes up the remaining 1/3 [6]. These two sections clamp down on the mRNA strand like a jaw or sandwich bread, then attract another RNA molecule named transfer RNA (tRNA) which lodges itself in the ribosome’s top section [6]. This tRNA brings with it amino acids for the creation of the protein and aids in translating [6]. The ribosome attaches to a section of the mRNA called the start codon [6]. The start codon is a 3 molecule structure making up part of the total mRNA strand (so, the entire mRNA strand is made up of multiple codons, 3 + 3 + 3 + 3, etc.) [6]. Each codon of the mRNA strand corresponds to a particular amino acid (presented by the tRNA) – example, A-G-C codon corresponds to the attachment of the amino acid serine [6]. Once the start codon is translated to an amino acid, the ribosome, and the rRNA within it (which aids in synthesis directly, as well as offering support for the protein being synthesized), moves down the mRNA to the next codon to attach the next amino acid to the developing protein until it reaches the stop codon, which indicates the protein strand is finished [6].
 

How does mTOR affect mRNA translation, exactly?

mTOR plays two primary roles; one, through the release of the aforementioned “brake” on protein synthesis by binding 4E-BP1, and in turn, leading to greater activity of eIF4E. Greater activity of eIF4E is important, because it is the initiation factor that initiates protein synthesis by moving ribosomes to the correct start codon on the mRNA strand via the recruitment of another initiation factor, methionyl-tRNA (mtRNA), designed to recognize start codons [7].

Secondly, mTOR is a regulator of mRNA elongation; this entails the process of moving a tRNA from the A site of the ribosome to the P site of the ribosome [3][7]. This is important, because once the initial tRNA has its amino acid translated to part of a protein, the ribosome must move down the mRNA to the next codon; in doing so, the two sites within the ribosome which house tRNA (sites A and P, or 1 & 2 respectively) must move their tRNA to the next slot in the ribosome (for example, tRNA in site A/1 slides to site P/2, and the tRNA in site P/2 is released from the ribosome – this allows a free slot, site A/1, for the next incoming tRNA)[8]. This process of elongation allows the protein to be fully synthesized as each tRNA attaches until the ribosome reaches the stop codon.

mTOR plays a crucial role in elongation as it interacts with S6 kinases 1 & 2, and in doing so, allowing both s6 kinases (but, especially s6 kinase 2) to phosphorylate eukaryotic elongation factor kinase (eEFK), which would normally bind to a protein, eukaryotic elongation factor (eEF) [7][9]. This protein is an accelerator of elongation by being involved in the translocation step in which the ribosome moves along the mRNA strand [7]. The binding of S6 kinases to eEF kinase reduce the amount of eEF kinase is bound to eEF, increasing the amount free to interact with the ribosome [9]. eEF, when bound by GTP, binds and delivers tRNA to the A site of the ribosome; when the correct tRNA is matched with the codon for translation, the GDP is hydrolyzed and the eEF then dissociates from the ribosome [10]. Interestingly, S6K may also phosphorylate a region on the ribosome, S6, but conflicting evidence keeps the result unclear – does it increase or decrease protein synthesis? Unsure [3].

S6K has several functions, and not all will be detailed here, but one of the final functions, related to protein synthesis, is related to transcription of mRNA. When mRNA is being transcribed from DNA, specific protein complexes, called spliceosomes, then splice the genetic sequence and eliminate “junk” DNA from the middle sections (introns) of the forming mRNA strand [11]. To be clear, introns, although thought to be useless for a time, do serve some specific functions like micro-autoregulation of genes, among other functions not necessary for this article [14].  The outer sections (exons), which are characterized as functional genetic material, are then reintegrated as exon junction complexes to create the nucleotide strand of the mRNA [3]. 20 nucleotides away from the exon junctions, the spliceosome also deposits exon junction complexes (EJC) that aid in the recognition of incorrect genetic material [3][12]. The discovery of improper genetic material is done by a series of proteins (surveillance factors) on the exon junction complex that recognize when an mRNA strand does not have its stop codon placed on the final exon junction – meaning, the stop codon is located at a location before its appropriate location (aka, premature termination codon), and the formation of a useless protein will be made if something is not done to intervene [11][12]. Ribosomes run into the stop codon prematurely, they signal to the exon junction complex, which undergoes a series of conformation changes, and eventually triggers mRNA nonsense mediated decay (aka, degradation) and the destruction of the ineffective mRNA strand [11][12]. After the first pass (called the pioneering round), the exon junction complex dissociates from the mRNA strand [11][13].

This quality assurance of the exon junction complex allows for more functional proteins to be created, but also speeds up the process of protein synthesis on the initial pass by the ribosome [3]. S6K plays a role in this as it binds, along with other proteins, to the exon junction complex, becoming part of the complex [11][13]. I should note that while S6K is a key piece to protein synthesis, it plays more of a role in enhancing protein synthesis, but its inexistence does not eliminate protein synthesis [3].

Realizing Protein Function & Protein Folding

At this point, the protein, or more appropriately named, polypeptide (multiple peptides/amino acids) has been created, extending from the ribosome. However, this polypeptide is useless without it being put together, like a cabinet that arrives disassembled from the store needs to be assembled to be useful. So, the cell has a few mechanisms by which to make a protein useful.

The first such mechanism is through a “chaperone” system as chaperone proteins (aptly named) attach to the polypeptide and twist and pull to manipulate the way the polypeptide is organized; so, instead of being a long chain, it is twisted into a functional conformation (ex, the shelf is put together by these chaperone proteins) [15]. This folding system is also useful for transporting proteins to various organelles. A chaperone will attach, but may not completely fold the polypeptide right away, but may keep part of it unfolded so as to allow it to fit through the nucleus through transmembrane relocation and introduce it to the correct location within the cell (mitochondrial proteins go to the mitochondria, for example); there, the chaperone finishes the folding of the polypeptide to a functional protein [15].

Here, a chaperone protein attaches to a polypeptide chain, rearranges it to its correct conformation and leaves, effectively finishing the creation of a functional protein for the cell, and to a larger extent, the body.

Another mechanism for protein folding into its appropriate 3D conformation is through the use of specific enzymes that break covalent bonds, restructure the protein conformation, and reapply covalent (electron sharing) bonds between different sections of the protein [15]. Other enzymes, however, simply cleave newly created proteins into subsections for ease of transport, but also to create new protein conformations that would otherwise not be possible by simply twisting the structure of the polypeptide [15].

Other proteins are glycosylated, meaning they are attached to carbohydrate molecules or chains of molecules, which change their look and function. The same can happen with lipids, and even a combination of carbohydrate molecules and lipids, called glycolipids [15].

In terms of how these chaperones, enzymes, and the like are able to conform polypeptide chains to functional proteins, it comes down to different bond principles, between covalent, hydrogen, hydrophilic and hydrophobic properties, and others [16]. There are four levels of structure that determine the look and function of a protein, but as those require a greater depth of chemistry, we will leave it at that.

Author: Nicolas Verhoeven
References

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[2] Proud, C. G. (2007). CELL SIGNALING: mTOR, Unleashed. Science, 318(5852), 926-927. doi:10.1126/science.1150653

[3] Sengupta, S., Peterson, T. R., & Sabatini, D. M. (2010). Regulation of the mTOR Complex 1 Pathway by Nutrients, Growth Factors, and Stress. Molecular Cell, 40(2), 310-322. doi:10.1016/j.molcel.2010.09.026

[4] Perl, A. (2015). Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases. Nature Reviews Rheumatology, 12(3), 169-182. doi:10.1038/nrrheum.2015.172

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[7] Wang, X. (2006). The mTOR Pathway in the Control of Protein Synthesis. Physiology, 21(5), 362-369. doi:10.1152/physiol.00024.2006

[8] Translation: Elongation. (n.d.). Retrieved from http://utminers.utep.edu/rwebb/html/translation__elongation.html

[9] Ruvinsky, I., & Meyuhas, O. (2006). Ribosomal protein S6 phosphorylation: from protein synthesis to cell size. Trends in Biochemical Sciences, 31(6), 342-348. doi:10.1016/j.tibs.2006.04.003

[10] Sasikumar, A. N., Perez, W. B., & Kinzy, T. G. (2012). The many roles of the eukaryotic elongation factor 1 complex. Wiley Interdisciplinary Reviews: RNA, 3(4), 543-555. doi:10.1002/wrna.1118

[11] Nott, A. (2004). Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. Genes & Development, 18(2), 210-222. doi:10.1101/gad.1163204

[12] Leblang, C., & Jurgens, D. (n.d.). Exon Junction Complex. Retrieved from http://biology.kenyon.edu/BMB/jmol2014/Magoh/EJC.html

[13] Ma, X. M., Yoon, S., Richardson, C. J., Jülich, K., & Blenis, J. (2008). SKAR Links Pre-mRNA Splicing to mTOR/S6K1-Mediated Enhanced Translation Efficiency of Spliced mRNAs. Cell, 133(2), 303-313. doi:10.1016/j.cell.2008.02.031

[14] Chorev, M., & Carmel, L. (2012). The Function of Introns. Frontiers in Genetics, 3. doi:10.3389/fgene.2012.00055

[15] Cooper, G. M. (2000). Protein Folding and Processing. In The Cell: A Molecular Approach(2nd ed.). Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK9843/

[16] Lodish, H. (2000). Hierarchical Structure of Proteins. In Molecular Cell Biology (4th ed.). Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK21581/

 

RNA polymerase is found in the nucleus of the cell, where DNA can also be found. DNA is split, used to synthesize RNA, then stitched back together. Now, we need only worry about RNA.

These images are showing the same process. Ribosomes clamp down onto the RNA strand and tRNA bring in the necessary amino acids for the ribosomes to read and stitch together.

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