Spell Checking Boiolgy

Original paper: Kinetic Proofreading: A new mechanism for reducing errors in biosynthetic processes requiring high specificity


Cells are sacks of chemicals that, through the trials and tribulations of evolution, have gained the ability to read information from their environment and then produce an output that assists in the larger organism’s survival. Mis-handling this information can lead to cell malfunction, mutation, or death, so it is important to understand how this works and how often it doesn’t. Using simple thermodynamic calculations, error rates are estimated to be thousands of times higher than they actually are (lucky for us!). Fundamentally, cells must obey the laws of thermodynamics, so some unknown intermediate process(es) must be dramatically reducing the number of errors in information processing. The question then becomes, what is that process? In a classic paper from 1974, J.J. Hopfield gives us an answer in a process he called kinetic proofreading. In doing so, Hopfield introduced the field of biophysics to the fundamental trade-offs that cells must make between using energy, accurately making a decision and the speed with which decisions can be made.

Before getting into kinetic proofreading, let’s get a better feel for our process of interest: protein synthesis. The central dogma of molecular biology can be summarized as DNA → RNA → Proteins. For the sake of simplicity, we will focus a bit more on the RNA → Proteins part. Like DNA, RNA molecules are long polymers that can be written down (or coded) as a simple sequence of letters. What’s really important is each three letter group called a codon. As the name suggests, each codon is a three-letter code associated with a specific amino acid [1]. When chained together in the order dictated by RNA, amino acids form a protein. Proteins then go on to perform almost every biological function you can imagine.

The RNA and the proteins are the input and output for our simplistic thermodynamic error estimate above (the one which predicts too many mistakes). Well, it turns out that this picture isn’t quite complete — there is also an emissary between the RNA and amino acids called “transfer” RNA, or tRNA. The tip of the tRNA directly binds onto the right amino acid, holding it in place as the growing protein gets formed.

It is extremely important that tRNA can (1) bind the right amino acid and (2) hold on to it for long enough to build the protein. Let’s call the tRNA binding site c and the amino acid X. When c and X meet, they create a combined unit cX, which is then produced into a protein. This can be written as the following reaction equation:

c + X \underset{k_{on}}{\overset{k_{off}}{\leftrightharpoons}} cX \overset{W}{\rightarrow} protein.

Let’s step through it. It says that X and c combine at an on-rate kon to form combined product cX, which we can think of as the amino acid attached to the tRNA molecule. cX can then either break apart at an off-rate koff, or it can go on to create the protein at a rate W. It turns out that kon doesn’t vary much for different amino acids, but the off-rates do. Experiments have measured that tRNA bound to the correct amino acid has a lower off-rate, giving more time for it to be produced into the correct protein. While tRNA unbinds a wrong amino acid faster, it still might go on to make a protein by accident. You can think of the wrong amino acid as being more “slippery” than the right one — tRNA can grab either one, but it is harder to hang on to the wrong amino acid. Using these differences in off-rates, one can estimate what the expected error fraction would be for proteins. The problem is that this estimate is way bigger than measured error fractions! Hopfield hypothesized that there must be something actively happening to close the gap. He named the exact mechanism he came up with kinetic proofreading.

The key to kinetic proofreading is to extend the time that the amino acid and the tRNA stay bound. This way, the tRNA is more likely to let go of the wrong, “slippery” amino acid, but hang on to the right one. Hopfield proposes putting an intermediate step between cX and the product. However, to be actually useful, going into the intermediate step has to be irreversible. To make something irreversible means to break time-reversal symmetry, which requires the consumption of free energy, usually in the form of “burning” ATP, the fuel used by cells. By burning this fuel, a slightly different form of cX is created, let’s just call it cX*. This new combined product is the one that then goes on to make the final product.  By adding this new intermediate step — the act of using up ATP to create cX* — it is possible for the amino acid to stay bound to the tRNA for twice as long as without the intermediate step. You get to run the process of discriminating between right and wrong amino acids twice, decreasing the error fraction significantly.

This argument that spending free energy can increase the accuracy of synthesizing proteins, has become a staple in understanding biophysics. Cells operate out-of-equilibrium by consuming energy and therefore are able to accomplish tasks much more accurately. Another trade-off is that the decision to be made, i.e. making the right protein, is done more slowly. These energy-speed-accuracy trade-offs are essential not only for protein synthesis but also DNA replication and triggering immune responses by T-cells recognizing foreign invaders. Equilibrium is death. By consuming energy from our surroundings, we are able to fend off the onslaughts of entropy and remain alive.


[1] There are approximately 20 amino acids, and there are different codon sequences that code for the same amino acid. ^

 

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