Life's Ratchet: How Molecular Machines Extract Order from Chaos

by Hoffmann, Peter M.

  An interesting suggestion, but this theory only became widely accepted when Walker solved the structure of the F1 part of the synthase. F1 is the part Boyer identified with the dial—it performs the actual ATP synthesis. F1 consisted of three identical units (each in turn consisting of two subunits, a and b) arranged in a circle like petals on a flower. Moreover, each identical b subunit had an ADP/ATP binding pocket. This circular arrangement was highly suggestive of rotary motion. But this motion was not proven until 1997, when a stunning experiment directly visualized the rotation of ATP synthase using fluorescence methods. Biochemist Hiroyuki Noji and his coworkers, then at Tokyo Institute of Technology, attached a short fluorescently labeled actin filament to the top of the F1 unit of a single ATP synthase. When the researchers fed the synthase with ATP, the machine ran in reverse, breaking down ATP into ADP and using the energy of ATP hydrolysis to fuel the rotation. This rotation then spun the attached actin filament around, like a majorette spinning a baton. Observing the actin filament in their microscope, Noji and colleagues saw that the F1 unit rotated clockwise at a pretty good clip, up to four rotations per second. Moreover, the generated force was appreciable—by their estimates as much as 45 pN.

  The efficiency with which the machine could turn energy from ATP hydrolysis into a rotation was astounding. In 2000, Noji’s group found that the machine was at least 88 percent efficient. Such a high efficiency is unheard of for macroscopic machines, but it gives us an idea of how the human body can be so efficient overall. We are based on tiny nanomachines—and only a nanomachine can be this efficient!

  Noji’s experiment proved that the ATP synthase was a rotary machine, but the researchers had run the machine in the opposite direction of how it would be used in mitochondria. They had seen that a clockwise turn broke down ATP. It seemed obvious that a counterclockwise turn would achieve the opposite: reattaching phosphates to ADP. But this was just conjecture until experimentally proven. Unfortunately, it turned out to be difficult to observe ATP synthase in its usual environment in the mitochondria of a living cell. Once you remove the synthase from the cell, it is removed from the rest of converting machinery. Thus, the proton-motive force to drive the motor cannot be established and the motor cannot be driven in the direction that converts electrical into chemical energy.

  Finally, in 2004, Noji’s team found a way. Instead of letting the protons do the work, they attached a magnetic bead to the center of the F1 unit of the synthase. Then, using a rotating magnetic field, they forced the magnetic handle either clockwise or counterclockwise. In other words, they hand-cranked the motor. When they cranked it clockwise, the amount of ATP in the surrounding solution decreased. The F0 unit acted as an ATPase, splitting off phosphates from ATP and releasing ADP. This confirmed their previous experiments. However, when they cranked the unit counterclockwise, ATP increased. The motor was now making ATP out of ADP.

  The F1 unit is coupled to an F0 unit, which is stuck in the membrane of the mitochondria. Unless cranked by some other motor, the F1 unit will never synthesize ATP, it will only break it down. It needs to be coupled to a motor that cranks it in the counterclockwise direction—opposite the rotation during ATP breakdown. In our cells, this motor is contained in the F0 subunit. The F0 unit consists of several subunits, but overall, it consists of a ring of identical c-subunits, a large a-subunit, and two b-subunits, which reach up to hold the F1 unit in place on top of the F0. Attached to the c-subunit ring is a flexible shaft (subunit γ), which transmits the rotation of the F0 unit to the F1 unit (Figure 7.11). The γ-subunit is asymmetrical. As it rotates, it alternately pushes on the b-subunit catalytic domains and changes the shape of the catalytic pocket to initiate the different stages of ADP to ATP processing. In other words, the catalytic site is adjustable to either accept ATP binding, ATP hydrolysis, or ADP release. The rotating shaft deforms each unit in turn to perform these steps in a sequential manner (Figure 7.12).

  FIGURE 7.11. The ATP synthase molecular electromotor and recharging station consists of the F0 electromotor, which is embedded in the mitochondrial membrane, and F1, the rotary ADP recharging enzyme. F0 consists of ten to fifteen c-subunits (depending on the organism) and an a-subunit and two b-subunits, which act as the stator. F1 consists of three a-subunits and three b-subunits. The catalytic sites are on the b-subunit and are modified by the rotating shaft (γ-subunit) to perform the different steps of the ATP synthesis.

  The F0 motor is a biological electromotor. The voltage created by the electron transfer chain forces protons to flow back across the membrane. However, they first have to flow through the F0 motor. The c-subunits of the motor have sites that accommodate protons. As protons enter the membrane, they are attracted to these sites and stick there. This site interacts with a hydrophobic patch in the membrane, which repels the proton. This creates a tilted energy landscape, and by a Brownian ratchet mechanism, the motor rotates. This brings another c-subunit into a position to load a proton, and the cycle continues. As the F0 unit rotates, each proton makes an almost complete circle before it arrives at a place where it is released and continues its journey across the membrane. When this motion is coupled to the F1 unit of the synthase, the F1-ATPase is run in reverse and becomes an ATP synthase, attaching phosphates to ADP.

  FIGURE 7.12. The F1 unit of ATP synthase seen from above, as the γ-subunit, or shaft, rotates counterclockwise. The shaft deforms the binding pocket in the identical three b-subunits such that at different times, either ADP and P bind, ADP and P are combined to ATP, or ATP is released. For every 360-degree rotation, this machine produces three ATP molecules.

  GETTING AWAY WITH LESS

  The F0 subunit structure was even more difficult to decipher than the F1 subunit structure. From early measurements, it was believed that four protons were required to rotate the machine 120 degrees and therefore produce one ATP molecule in the F1 catalytic unit of the synthase. This would require the F0 unit to contain 4 × 3, or 12 subunits (called c-subunits) that drive motion when protons pass through. However, studies of ATP synthases in different organisms (ATP synthases are ubiquitous in everything from plants to amoeba to humans) showed that there was a variable number of c-subunits, depending on the species. Plants appeared to have 14 c-subunits, cyanobacteria 13–15, certain other bacteria 11, and, finally, yeasts and the bacterium E. coli (and, presumably, humans) 10. Since it is believed that each subunit uses one proton to initiate a rotation, this variable number of c-subunits suggests that there are variable numbers of protons needed to synthesize one ATP molecule. For example, if 14 c-subunits are present in plants, this would suggest that 14/3, or 4.7, protons are needed to make one ATP, but this does not seem the case from experiments. In plants, only 4 protons are needed. At the other extreme, 10 c-subunits would translate into 3.33 protons per ATP molecule, suggesting an improvement in efficiency. But this has not been proven, either. Many mysteries remain.

  Twisting: The DNA Machines

  A detailed discussion of the entire machinery that reads, translates, repairs, duplicates, and maintains DNA would easily fill an entire book. But for the purpose of showing how molecular machines are used in almost everything our cells do, a brief overview will suffice. The DNA in our cells is rolled up—and then the rolls, in turn, are rolled up again—into compact DNA-protein structures called chromosomes. This packaging of DNA into chromosomes is performed by molecular machines.

  DNA contains the information both to make proteins and to regulate the making of proteins. It does not contain the information of how to make a human—at least not directly. There is no gene for a toe or an eye. There are genes to make protein components of toes and eyes. The actual development of a human is a complicated process and involves the accurate timing of the synthesis of many proteins, their interaction, their regulation, and the movement of molecules by molecular motors. Many of these immensely complicated processes are still not well understood, although we now have a much better understanding than we did just twenty years ago. One of the
se well-understood processes is how a protein is made according to the instructions contained in DNA.

  DNA is a double-helix molecule with a backbone made of alternating sugars (deoxyribose, which gives DNA its D) and phosphates. It looks like a twisted ladder. The rungs of the ladder are composed of various combinations of four nucleotide bases that constitute the DNA alphabet. The bases are adenine (A), guanine (G), thymine (T), and cytosine (C). Adenine is the same molecule that is part of adenosine triphosphate (ATP). The two sides of the double helix are complementary—this means that an A on one side always pairs up with a T on the other, and a G will always pair with a C. This specific pairing is achieved by hydrogen bonds, which are placed in such a way that, for example, T can form hydrogen bonds only with A, but not with G or C (Figure 7.13). Hydrogen bonds on their own are relatively weak, but the large number of hydrogen bonds fastening the two helices together gives great strength to DNA.

  There are multiple advantages to DNA’s double-helix structure with complementary base pairing. For one, the redundancy provided by the pairing of bases allows cells to repair DNA when it is damaged. Second is the molecule’s ability to replicate itself, as James Watson and Francis Crick stated in their famous paper: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” In other words, if you want to copy DNA, all you’d have to do is open up the helix and float some free bases around, and they will automatically pair up with their correct counterparts. Before long, you have made two helices out of one, both perfect copies of each other. It’s not quite that easy, as we will see, but that’s the basic idea.

  FIGURE 7.13. DNA structure. On the left is the overall double-helix structure. The two helices are made of alternating sugar and phosphate groups, while the rungs connecting the two helices are pairs of complementary nucleotides (A [adenine], C [cytosine], T [thiamine], or G [guanine]), as seen in the magnification on the right. The specific sequence of these nucleotide letters encodes the DNA message.

  The language of DNA uses three-letter words made up of four different letters (the bases A, C, T, and G). The three-letter word AAC, for example, codes for a specific amino acid in a protein. AAC happens to code for asparagine, while CUU codes for leucine. These three-letter words are called codons. The set of all three-letter words is the genetic code. A gene is a DNA sentence, a string of codons, which encodes a complete amino acid chain for a specific protein. For example, the KLC1 gene on chromosome 14 encodes the protein sequence for kinesin-1, our waddling motor protein.

  The making of a protein proceeds through two distinct steps: transcription and translation. During transcription, the necessary information to make the protein is transcribed to another information carrier, RNA, which is a close relative of DNA. Why is this intermediate step needed? The DNA in our cells is kept safe in the cell nucleus. We do not want to cut out and remove parts of the DNA each time the cell manufactures a protein. Instead, the cell makes a temporary information carrier made of RNA, which can leave the cell nucleus and carry the information to where it is needed. This RNA information carrier is fittingly called messenger RNA (mRNA).

  Thus, there are three major processes involving DNA in our cells: copying DNA when the cell divides (replication), copying DNA to RNA when a protein is to be made (transcription), and packaging DNA into chromosomes.

  THE COPY MACHINERY

  DNA is a very stable molecule. This is a good thing, as we do not want anything to happen to our DNA. Mistakes in our DNA can have disastrous consequences, such as the development of cancer. However, if the molecule is always curled up into a rigid structure, it would be impossible to copy or transcribe DNA. Therefore, we have to unravel, open, read, copy, and close it up again. DNA is copied when cells divide (each cell receives a complete copy of the genetic material), and it is transcribed each time a cell needs to make a protein. Although these processes seem superficially similar, they work in completely different ways.

  FIGURE 7.14. The DNA replication machinery. Helicase untwists the helix and separates the two strands. Topoisomerase cuts and re-splices the DNA to avoid tangles. Polymerases copy the DNA and make a new double helix out of each separate strand. One of the polymerases follows the helicase, while the other two work in the opposite direction. Thus they are forced to copy the DNA in fragments, which are spliced together by ligase.

  During replication, a large number of molecular machines and enzymes work together to make sure the DNA is not damaged and that a true copy of the original is produced (Figure 7.14). But there are several complications. First of all, the DNA strand needs to be opened up. Second, if you have ever tried to untangle a twisted telephone cord, you can imagine that opening up a double helix can create a mess: Without proper precautions, the strands would quickly twist into a knot. To avoid a tangled mess, a molecular machine called topoisomerase snips one of the strands of DNA from time to time and passes it through the other to compensate for the final unwinding of the two strands by another molecular motor, helicase.

  There is another complication: The two complementary strands of DNA are oriented in different directions. Like proteins (which have N- and C-terminals), DNA has different ends as well. The so-called 5' end has a phosphate group, and the 3' end has a sugar group. One of the strands runs from 3' to 5', while the other runs from 5' to 3'. Unfortunately, the machine that copies the DNA can only run in one direction: from 5' to 3'. This is fine for one of the two strands, because this copy machine, called DNA polymerase, can follow the machine that opens up the DNA (helicase). But the other strand points in the opposite direction. This means the copy machine on this strand has to move away from the point where the DNA is being split. What’s worse, while this is happening, more DNA is split behind the machine. How to solve this problem? Copy a small section of the DNA, move the copy machine back, copy another small section, splice the sections together using another machine (called ligase), and so on.

  TWISTING THE HELIX

  Although space prevents us from discussing every molecular machine in DNA replication, we will concentrate on helicase, the machine involved in untwisting (and opening) the DNA helix. These untwisting machines have two jobs: separating the complementary DNA strands, and untwisting them to get them ready for copying. Untwisting the two separated strands can lead to more twisting in the not-yet separated DNA. Topoisomerases make sure the helicase motors do not turn the DNA into a tangled knot. Structurally, helicases look like a six-sided star with a hole in the middle through which DNA passes. How is the DNA spooled through the middle? Helicases self-assemble around the DNA, most likely by opening the six-sided helicase ring, letting the DNA in, and closing up again. The six-sided shape may help the helicase continue to do its job while the DNA curls around as it moves through the ring center.

  As discussed, the rungs of the DNA ladder—the complementary bases—are held together by hydrogen bonds. Studies have shown that helicases break these bonds much faster than these bonds would break on their own. Helicases are, first of all, enzymes that speed up the splitting of the DNA double helix. Remember, any bond will eventually break on its own. Every molecule is incessantly bombarded by the molecular storm— so there is always a possibility that, by chance, the bond will receive enough energy from the thermal chaos to break. The average time this takes depends on the height of the activation barrier. Helicases lower the activation barrier for splitting the two DNA strands by manipulating the charge on DNA and therefore speeding up the unzipping process. But helicases not only facilitate splitting the DNA; they also move along DNA like a pair of scissors. As they cut, they move forward, with the motion fueled by ATP. Helicases are therefore molecular motors as well. How this motion works is still being debated: Is it a tightly coupled power stroke or a Brownian ratchet mechanism? This debate may sound familiar. Some research suggests that ATP binding weakens the helicase’s bond to DNA, allowing the helicase to move relatively freely. This would support
a Brownian ratchet mechanism.

  SENDING THE MESSAGE

  The DNA in our cells contains all the information to make every protein needed by our bodies. To turn DNA instructions into actual proteins, the instructions have to be copied first to an RNA messenger, which transports the instructions to the factory floor, where the machines that translate DNA instructions into proteins are located. RNA is a more flexible and more ancient cousin of DNA (many people believe that life started with RNA). RNA contains a different sugar—ribose, as opposed to the deoxyribose in DNA—in its backbone, making it more flexible than DNA. Like DNA, RNA has four bases, three of which—A, C, and G—are the same as in DNA. However, the fourth base in RNA is uracil (U).

  Because RNA also has four different bases, DNA language is easily transcribed into RNA language (simply replace the T with a U). This allows RNA to play the role of messenger whenever the cell consults its DNA library to make a protein. The transcription of DNA into mRNA is accomplished by a molecular machine called RNA polymerase. This machine starts working when it encounters a specific DNA sequence, or promoter. The promoter marks the spot where the RNA polymerase is supposed to start transcription. Always the same, the promoter consists of two parts, ten and thirty-five base pairs before the sequence to be translated. Once the polymerase finds the promoter, it attaches to the DNA and starts making an RNA copy of the information it finds. RNA polymerase is the Swiss army knife of molecular machines. While DNA replication requires an army of enzymes and molecular machines, RNA polymerase works on its own—opening up DNA, transcribing it, moving along DNA, proofreading, and finally terminating the copying process.