Please welcome this month's Scicurious Guest Writer, Abid Javed! Not only did he write his post, he also drew some of his own art!
Machines can be large and complex. Take a car, for instance. It has an engine that allows it perform the task of driving us humans from one place to another. A single misstep or damage to one of many car parts and the machine would stop working all together. For example, a rusted car engine would prevent the car from starting, let alone moving it forward. Now consider this machine idea in biology. Just like their man-made counterparts, biological machines can be complex and large, and can perform tasks with tremendous power. ATP Synthase, for example, is a large protein machine in cells that functions by rotating itself to power its ATP (energy) molecule production. Similarly, we have ribosomes as the protein-making (translating) machines in cells. Either functioning freely in the cytoplasm or embedded within the membranes in compartments of the cell, these machines work tirelessly to make new proteins. Ribosomes in the cytoplasm of the cell are like cars, driving along the messenger RNA strand (mRNA), trailing the growing protein chain with it until it reach its final destination stop (the stop genetic code on mRNA) to finish protein synthesis (2). Found in all living organisms that make proteins, how does this machine achieve its mighty feat of a role?
Structure of a ribosome
Let us first consider the structural composition of this biological machine. It is assembled from two main body parts - one small, 30S (S is a Svedberg unit used to indicate the weight of molecules) subunit, and one large 50S subunit. Each of these subunits is made up of ribosomal RNA and protein molecules that complement each other in achieving ribosome’s function of protein synthesis. The large, 50S ribosome subunit has a carefully folded RNA molecule (16S ribosomal RNA), interwoven between the 50S ribosomal proteins. On the other hand, the small 30S subunit has two RNA molecules (23S and 5S ribosomal RNA) with its ribosomal proteins encasing it (2), (3). The individual small and large subunit parts are synthesized inside the nucleus of a cell that, once in the cytoplasm, assemble into one 70S ribosome unit (2).
Like a car, a ribosome machine has purposeful sites designated within its structure. Inside many older cars, for example, there are the front driver and passenger seats, with small middle seat sandwiched between the two. Similarly inside the core of the ribosome, you will find three sites where the main action occurs. First, there is the driver A site (aminoacyl-tRNA site), that allows the entry of new tRNA molecules attached to an amino acid. Next to it is the middle passenger P site (peptidyl-tRNA site), that holds a growing protein chain and the intermediate tRNA molecule in place. And finally, the front passenger E site (the exit site) allows the exit of both the used tRNA molecules and the newly made protein chain out of the ribosome. Essentially, the small subunit part of the machine is for recognizing and correctly binding the incoming tRNA molecules and the large part is for facilitating the protein synthesis action (fig. 1).
(Source, by Darrell Sharp)
One of the reasons why the ribosome is so big is because it has a large substrate to accommodate; the tRNA molecule. The tRNA molecule is folded into L-shaped RNA structure from its genetic sequence. On one end of its structure, it carries the attached amino acid with it and on another it exposes a three letter genetic code (anti-codon to mRNA’s codon) that acts as a specific recognition site for binding to the mRNA molecule inside the ribosome. Essentially, a tRNA molecule functions as a cargo-like molecule during the protein translation event by bringing in a new amino acid into the ribosome for it to be incorporated into the growing protein chain (2). Once it has finished its job inside the ribosome, the tRNA molecule recycles itself by scavenging for new amino acids that it can bring back for another round.
Many machines need accessory parts to function and the ribosome is no exception. During the protein synthesis process, the ribosome machinery is aided by three accessory proteins that help the ribosome in translating proteins effectively. EF-Tu (elongation factor thermo unstable) protein allows the ribosome to bind a specific tRNA molecule in it’s A binding site to begin translation (5). EF-G (elongation factor G) proteins help the ribosome shuffle both the tRNA and mRNA along during the protein synthesis step (6). The release factor protein (RF1) eventually helps the ribosome to facilitate the exit of both tRNA and newly synthesised protein chains from the exit site.
The ribosome at work
As soon as the ribosome has assembled all of its individual parts to make a single bodied unit, it comes alive as a machine to makes proteins. It does this by combining the work done by the individual small and large subunits in gathering the tRNA-bound amino acid components from the cytoplasm and putting them together to make extensive protein molecules. There are three stages in this entire process, occurring at the three respective sites inside the machine. After the free ribosome has assembled and is bound to the mRNA strand in the cytoplasm, the first step is to allow specific entry of an incoming tRNA molecule at the A site of the small ribosomal subunit. On this subunit’s ribosomal RNA strand, there are two genetic gatekeeper groups that act as guards, only allowing specific tRNA molecules to bind. This is through their specific chemical interactions with the incoming tRNA molecule that either allows the entry in or simply rejects it. The ribosome certainly cannot allow for non-specific binding as that will disrupt the machines mechanics (4). An engine like a ribosome (comprising of both the substrate binding small subunit and protein-making large subunit) needs fuel to function and this comes in the form of quick-energy releasing molecules called GTP (Guanosine triphosphate). These are carried by the accessory proteins (EF-Tu and G proteins) that eventually fuel the tRNA binding and movement along the A to E site inside the ribosome during the protein synthesis event (5), (6).
Once the correct tRNA (carrying the amino acid) is bound to the mRNA strand inside the ribosome at the A site, the machine gets ready to start putting the protein molecules together by bonding the incoming amino acids inside it. The ribosome machine has a special, flexible ability to move and wrap itself around the bound tRNA molecule once it binds, so that it can make the bonding of the accompanied-amino acids easier by bringing them closer to each other (7), (9). With fixating the tRNA molecule correctly at the A site in the small subunit, the protein synthesis action at the large subunit begins.
Inside the ribosome machine’s large subunit part, the main chemical event of protein synthesis takes place at the A and P site junction known as the peptidyltransferase center, where the ribosome machine chemically bonds the individual amino acids sequentially at the designated ribosome sites so that the process is progressive and doesn’t come to a halt. Along with the accessory proteins (EF-Tu and EF-G and RF1 stated earlier), the tRNA and the amino acids hop from A to P to E site within the two ribosomal parts, allowing the protein chain molecule to grow progressively (8). Once the last tRNA molecule binds to its corresponding stop mRNA code in 30S machine part, the peptidyltransferase centre in the large subunit part realizes that it is time to stop. The protein is consequently terminated and assisted by the RF1 accessory protein and the freshly made chain slithers itself through the ribosome E site exit tunnel and out of the ribosome (3), (9), (10). Within this whole process, the job of the ribosome is not only to act as the ribozyme (to chemically make proteins) but to also act as a filtering machine so that it allows the correct incorporation of the amino acids within the growing protein chain and reduces protein translation error rate (10). Not having this filtering capability would otherwise cause the ribosome to incorporate the wrong amino acids within a protein, which would result in a completely dysfunctional protein being made. Hence, it is important that the ribosome is able to sustain its filtering characteristic.
Targeting the function of the ribosome.
However, with a big machine like ribosome working rigorously as it does, there is inevitably room for errors to occur. As mentioned earlier, a faulty machine part or convolution with its function would amplify the error producing rate of the ribosome. Scientists have considered this as a major step towards treating various pathological diseases by making the ribosome machine dysfuctional in making correct protein molecules. For example, current work is exploiting the machinery of the ribosome to create new drugs by inhibiting protein synthesis in cells, something that could be a powerful tool in stopping things like bacterial infections. Natural antibiotics and chemical inhibitors are a few examples that have shown promising results in inhibiting ribosome function in cells. The inhibitory action of these molecules directly results in the prevention of the large 50S machine part from functioning; weakening the peptidyltransferase centre’s nuts and bolts and therefore preventing protein synthesis in the ribosome. In essence, by inhibiting the protein synthesis process, it directly prevents the growth and survival of the pathological organisms that are being targeted and hence proves a useful strategy at the interface of DNA and protein molecules in the treatment of pathological diseases (10), (11).
All this hard work in understanding the molecular inner workings of this complex machine begs the question of why have we been so keen on it? Protein molecules are at the heart of living biology and understanding their fundamental synthesis process (by the ribosome machine) allows us to get to the bottom of questions relating to how proteins actually fold once they are synthesized, and what leads to them not adapting a correct shape (which can result in protein misfolding, a symptom that characterizes some neurological diseases such as Alzheimers). The sheer hard work on elucidating the ribosome machine’s behaviour was rightfully recognized and awarded the Nobel Prize for Chemistry in 2009 (12). There is still much more work to be done, and important things to be learned from the ribosome presently. But for all the work that remains, it is still easy to see what a remarkable biological machine a ribosome really is.
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2. Steitz A. Thomas. 2008. A structural understanding of the dynamic ribosome machine. Nature Reviews Molecular Cell Biology. Vol.9: 242-253.
3. Rodnina V. Marina, BeringerMalte and Wintermeyer Wolfgang. 2007. How ribosomes make peptide bonds. Trends in Biochemical Sciences. Vol.32: 20-26.
4. Wimberly T. Brian, Brodersen E. Ditlev, Clemons M. William, Warren-Morgan J. Robert, Carter P. Andrew, Vonrhein Clemens, Hartsch Thomas &Ramakrishnan V. 2000. Structure of the 30S ribosomal subunit. Nature. Vol. 407: 327-339.
5. Stark Holger, Rodnina V. Marina, Appel-RinkeJutta, Brimacome Richard, Wintermeyer Wolfgang & Heel van Marin. 1997. Visualization of elongation factor Tu on the Escherichia coli ribosome. Nature. Vol. 389: 403-406.
6. Rodnina V. Marina, Savelsbergh, Katunin I. Vladimir &Wintermeyer Wolfgang. 1997. Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature. Vol. 385: 37-41.
7. PapeTillmann, Wintermeyer Wolfgang and Rodnina Marina. 1999. Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome. The EMBO Journal. Vol. 18: 3800-3807.
8. Julian Patricia, Konevega L. Andrey, Scheres W. H. Sjors, Lazaro Melisa, Gil David, Wintermeyer Wolfgang, Rodnina V. Marina and Valle Mikel. 2008. Structure of ratcheted ribosomes with tRNAs in hybrid states. Proceedings of the National Academy of Sciences of the United States of America. Vol. 105: 16924-16927.
9. Valle Mikel, ZavialovAndrey, SenguptaJayati, RawatUrmila, Ehrenberg Mans, Frank Joachim. 2003. Locking and Unlocking of Ribosomal Motions. Cell. Vol. 114: 123-134.
10. Liljas A. 1999. Function is Structure. Science. Vol. 285 (5436): 2077-8.
11. Ochi Kozo, Okamoto Susumu, TozawaYuzuru, Inaoka Takashi, Hosaka Takeshi, Xu Jun, Kurosawa Kazuhiko. 2004. Ribosome Engineering and Secondary Metabolite Production. Advances in Applied Microbiology. Vol.56: Pg 155-184.
12. Venkatraman Ramakrishnan, Thomas A. Steitz, Ada E. Yonath. Nobel Prize for Chemistry, 2009 :- www.nobelprize.org/nobel_prizes/chemistry/laureates/2009/press.html
Abid Javed is an undergraduate Biochemistry student at the University of Manchester. After completing his degree this year , Abid will start his PhD studies with Dr John Christodoulou at UCL to tease apart the inner workings of the ribosome. Someday, he wants to hold an art exhibition to show how structurally beautiful proteins really are.