Molecular Machine Safari
From the safety of our imaginary tour vehicle, we drive inside a cell, looking at some of the elaborate robotic machines at work.
The Nano-Muscle Ratchet
Our tour starts by riding a vesicle into the cell. Behind us is a dynamin machine closing the vesicle. Dynamin is a molecular motor that plays a central role in clathrin-mediated endocytosis, a means of importing cargo from outside the membrane. As the vesicle forms, clathrin molecules interlock around our vesicle, creating what looks like a geodesic dome. We watch as dynamin grabs the neck of the vesicle behind us and tightens it like a balloon. Dynamin has 100,000 atoms! Oleg Ganichkin tells us why we humans rely on this motor working properly:
Dynamin is a mechanochemical GTPase that plays a central role in clathrin-mediated endocytosis (CME). The 100-kDa protein polymerizes into helical filaments that coil around the necks of vesicles budding from the membrane. In the presence of guanosine-5’-triphosphate (GTP), such filaments constrict, whereas GTP hydrolysis is necessary to cut the neck, thus allowing the vesicle to separate. This process is crucial for cellular nutrient uptake and for synaptic transmission. Mutations in dynamin are associated with severe neurodegenerative disease and muscular disorders. [Emphasis added.]
Using linkers between domains, dynamin wraps around the neck of the vesicle like a boa constrictor and tightens it. In their paper “Quantification and demonstration of the collective constriction-by-ratchet mechanism in the dynamin molecular motor,” in PNAS, Ganichkin et al. found that the constriction ratchet really puts the squeeze on the neck: “we quantitatively validate the prevailing constriction-by-ratchet model for nature’s strongest torque-generating motor: the dynamin ‘nanomuscle.’”
As our safari vehicle exits the vesicle, we watch the clathrin molecules dissociate for re-use. The tour now continues into the nucleus. The driver inserts our ticket into the nuclear pore complex for validation, then we enter a world of dazzling activity. He drives us to a gene being transcribed on a DNA strand. He points to a huge molecular machine called the spliceosome. It is grabbing the nascent messenger RNA (mRNA) at several points, cutting out segments. What’s going on? Karan Bedi, a narrator from the Center for RNA Biomedicine at the University of Michigan, comes over and explains:
Several processes take place to produce mature mRNAs that then can be exported to the cytoplasm and used as a template for protein synthesis. After initiation of transcription and the go-ahead of elongation to produce the pre-mRNA, introns need to be spliced out and the protein-coding exons connected. At first, pre-mRNA is made as a complementary sequence of the DNA but with slightly different chemistry and includes all the introns. Then the spliceosome machinery, made up of about 300 proteins, assembles “co-transcriptionally” at each intron junction as the RNA emerges from its synthesis. “Splicing is an incredibly complex process because of the great number of proteins involved that repeatedly need to assemble and disassemble at each junction. Also, the speed at which transcription generates RNA is quite fast so the splicing process has to be well organized. Many steps can go wrong and lead to various pathologies, which is why it is so important to have a better understanding of how splicing happens and how it is regulated,” said Bedi.
Bedi’s team found even more regulating factors that participate in this complex splicing process beyond the 300 protein parts of the spliceosome itself.
While in the nucleus, the driver takes us to the management office, where researchers from the Friedrich Miescher Institute for Biomedical Research (FMI) are inquiring about how the transcription factors work. A team member comes over and explains,
If the human genome were a company, transcription factors would be the top-level managers, controlling when and how much genes are turned on in specific cells. These proteins typically bind short strings of DNA called ‘motifs’. Scientists estimate that there are up to 2,800 transcription factors, but binding motifs have been identified for only about 800 of them.
The Swiss team learns that the transcription factors act like switches that bind to specific motifs. With that knowledge, they go “fishing” for one, using a CGCG motif as bait. They get a strike! They “detected the Btg3-associated nuclear protein (BANP) as the only protein bound to the CGCG motif.”
“This protein has been known before, but it was thought to repress gene activity at the periphery of the nucleus,” Grand says. “We show that it does quite the opposite: it’s a very potent activator of gene expression.”
“Why hasn’t this fish been caught before?” someone asks.
Despite its key role in regulating gene expression, BANP had been hiding in plain sight. “We believe that is because BANP is so essential: you touch it, and the cell dies,” Schübeler says. “This made it hard to identify it by any kind of genetic screening approach, which makes us wonder whether there are more of these factors out there that are invisible to us for the same reasons,” he adds.
The safari minivan follows the spliced mRNA out the nuclear pore into the cytoplasm. It drives on a microtubule to a working ribosome. There, we see a team from St. Jude Children’s Research Hospital observing and taking notes. They know that this is another crucial machine for life.
Expression of the information encoded in DNA makes life possible. DNA is first transcribed into RNA; RNA is then translated into protein. The ribosome is the molecular machine responsible for this second step. It decodes messenger RNA and makes proteins. Importantly, the ribosome is responsible for the synthesis of cellular proteins in all forms of life.
As they watch the mRNA chugging along through the ribosome at up to 20 bases per second, they marvel at the low error rate: one typo in 1,000 to 100,000 at this stage. Proofreaders down the line will reduce that error rate to one in ten million. The ribosome, furthermore, keeps that accuracy rate that high over thousands of cycles. The team moves their cryo-EM machines to take pictures of six new structures not previously described. They publish their findings in Nature, “Structural basis of early translocation events on the ribosome.”
The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life.
“Any references to Darwinian evolution in your paper?”, we ask. “Are you kidding?” one laughs.
The Fail-Safe Powerhouse
A tourist wants to visit the powerhouse of the cell, the mitochondrion. She saw Discovery Institute’s animation of ATP synthase and wants to see it in real life. Inside the double membrane, the passengers find a team from the Tokyo University of Agriculture and Technology investigating the organelle’s fail-safe mechanism. What happens, for instance, if the pH rises and all that power in the proton motive force goes into producing more reactive oxygen species (ROS), which are toxic to the cell?
Mitochondria are double membraned, with genetic information and functional units contained within its internal matrix. Mitochondria convert chemical energy into power for the cell by moving protons from outside to inside the matrix with the help of an enzyme responsible for energy conversion. But mitochondria also appear to impulsively and temporarily take up protons through another protein through a process called spontaneous transient depolarization.
Aha, the Japanese team found: increasing the pH increases the depolarization transients, and thus increases the number of ROS. And yet they know that “mitochondria have a mechanism to spontaneously avoid the production of excess reactive oxygen species,” so they want to figure that out. We leave them to their work as we marvel at the rotary engines in pairs spinning at 5,000 rpm up and down the cristae, generating fantastic amounts of ATP molecules. We watch as these energetic molecules are carefully ferried out the double membrane of the mitochondrion by automated robot-guarded gates, out to the cytoplasm to wherever they are needed. “This is amazing,” the female passenger exclaims. “It’s more wonderful than I had imagined!”
The driver must get back for the next group, so the passengers reluctantly take their seats and ride a microtubule out the exit. They chat about how terrific the tour was; they had no idea the cell was so complicated and yet orderly, too. They each vow to come back again for more. There is so much to see!