Irreducible complexity in the assembly of molecular machines

irreducible complexity

We know that many molecular machines are irreducibly complex (CI) in their functioning. Even more IC is the process of putting them together in the cell. A good example of this is the process of building our good old standby machine, ATP synthase (review our animation recognize the F0 rotating part and the F1 synthesis part).

A new tour de force from He et al. in the Proceedings of the National Academy of Sciences (PNAS), co-authored by Nobel Laureate John E. Walker (who, at 77, still researching these tiny rotary motors), describes new information about how these multi-part machines are put together . In an accompanying commentary on PNASThree scientists (Song, Pfanner and Becker) put it bluntly: “The assembly of mitochondrial ATP synthase is a complicated process that involves the coordinated association of mitochondrial and nuclear coded subunits. Here’s a taste of what they mean (don’t worry, it won’t be on the test):

Based on their findings [He et al.], they propose an elegant model of the construction of the membrane domain of human ATP synthase (Fig. 1, upper). In a branch, an F1–associated intermediary c-rings with the peripheral stem and the supernumerary subunits e and g. In the other branch, the F1 domain assemble first with the peripheral stem and the supernumerary subunits e, g and f. The two tracks merge into an intermediate key assembly which contains the F1 domain, the c-ring, the peripheral stem, and the supernumerary e, g and f subunits. In all these vestiges [i.e., incomplete] ATP synthase complex, the inhibitor protein IF1 is enriched to prevent hydrolysis of ATP by uncoupled ATP synthase. The presence of the supernumerary subunits e, g and f is crucial for subsequent integration ATP6 and ATP8 mitochondrial coded subunits which are stabilized by addition of 6.8PL. Thus, the proton conducting channel between ATP6 and the c ring is formed. TO at this point, ATP synthesis is coupled to the proton driving force and the inhibitory protein IF1 is released. Finally, DAPIT is added to the assembly line to promote dimerization and oligomerization ATP synthase. [Emphasis added.]

Whether or not you can keep up with the lingo is not as important as what they witnessed: an “elegant” process that requires precise timing and coordination. Different parts of the machine must arrive on time and assemble into intermediate (vestigal) shapes that are not functional on their own. An inhibitor protein ensures that the machine does not turn on earlier than expected. The proton conducting channel must be perfectly formed so that it does not “leak” protons. It is only when all the parts are ready that the machine begins to spin, but even then the job is not finished. Another actor is “added to the assembly line” to position the machines over the folds of the mitochondrial membrane (called ridges) at precise angles and spacings for optimum productivity.

The parts must arrive on site on time. Some of them come from the kernel, which must appear several miles away on the scale of the machine. Some are built locally by genes within the mitochondrial genome. Interestingly, there are differences between yeast and humans in terms of the genes encoded where and in what order they are assembled. But the proof of the pudding is in the breathing after eating: both versions of the machine work effectively for their respective bodies.

The intermediate structure, a bit like a scaffolding on which the machine will be built, is also irreducibly complex:

We have shown that the assembly of human ATP synthase in the inner organellar membrane involves the formation of a monomeric intermediate composed of 25 proteins encoded in the nucleus in which two mitochondrially coded subunits are inserted and then sealed by association of another nuclear coded protein, thus dimerizing the complex. The association of a final nuclear protein oligomerizes the dimers face to face along the edges of the ridges.

Note that the parts of the different genomes must work closely together. It’s like a manufacturing facility receiving parts locally and from India that must meet agreed specifications to match. There are also rules for importing, just like for parts arriving from a distant country. Nuclear coded coins must pass through two separate checkpoints (the inner and outer membranes of the mitochondria), each of which has their own robotic security personnel to validate them and facilitate their transport inward.

Previous work has shown how the completed machine “factory” is organized within the mitochondria. A specific nuclear protein seals them in two (dimers) at an angle, such as rotating F0 proton pumps can maximize the supply of proton fuel, while the F1 the parts, where ATP synthesis occurs, are more distant from each other so as not to encumber the output molecules. A “final nuclear protein” connects the dimers together (oligomerizes them) along the edges of the membrane. Longitudinal spacing is also tightly controlled, so they don’t crowd together. Each stitch of the assembly is directed by program. When all is done, rows of ATP synthase engines are arranged like turbines in a hydroelectric power plant, feeding on a stream of protons produced by machines upstream of the respiration transport chain.

Set of ribosomes

Spectators of cellular animations like those of Unravel the mystery of life One could never forget the assembly line process inside the ribosome, where precisely sequenced messenger RNAs are paired with transfer RNAs carrying amino acids to form proteins. The inlet tunnels for ingredients and the outlet tunnels for polypeptides, and everything in between, must be positioned exactly for proper operation. The ribosome is certainly one of the most amazing examples of information translation in all of nature. But how is the ribosome itself constructed?

Nature provided an early version of an unedited manuscript by Sanghai et al. on ribosome assembly. Although it has been accepted for publication, it will be subject to editorial revisions. The subject, however, appears to show another astonishing case of irreducible complexity in the construction of this important molecular machine. Here is the summary :

The first co-transcriptional events of eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, due to limited structural information, the role of these factors during early 60S nucleolar assembly is not fully understood. Here, we have determined cryo-EM reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions up to 3.4 Ã…. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and subsequent factor binding. This is accomplished through the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix domain proteins and their binding partners form a ring structure at the boundaries of the rRNA domains. to support the architecture of the maturing particle. The mutually exclusive conformations of these pre-60S particles suggest that the formation of the polypeptide exit tunnel is accomplished by different folding routes during the later stages of ribosome assembly. These structures rationalize the previous genetic and biochemical data and highlight the mechanisms leading to the assembly of eukaryotic ribosomes in a unidirectional manner.

The requirements of IC are met in this description: “a multitude of transient assembly factors” systematically regulate and refold the proteins that will be used to build the machine. The authors mention “21 ribosome assembly factors that stabilize and remodel” RNA and proteins before the machine is even operational. Inside the growing ribosome, a scaffolding holds the factors of the exit tunnel in place. Everything is choreographed in time and space with “mechanisms leading to… unidirectional assembly”.

Here we see many parts working together on a timeline. The parts alone do not work individually. You can have all the proteins delivered to the job site, and nothing will happen without the programmed mechanisms to put them together in order. Some parts hold others in place, others guide the folding of protein parts, and some even prevent premature assembly. All subdomain assembly paths are regulated by a master program, so that each group of steps follows a “one-way” plane to the finished product. It is a wonderful process of assembling integrated circuits that produces an integrated circuit machine. If five parts of a mousetrap are enough to indicate IC, what about dozens of parts, all in a programmatic assembly sequence?

In Unlocking, concerning the assembly of the bacterial flagellum, Paul Nelson described how the hierarchical integrated circuit of the assembly of the machine in the cell challenges the Darwinian theory. “To build this flagellar mechanism, or tens of thousands of other such mechanisms in the cell, you need other machines to adjust the assembly of these structures. And these machines themselves require other machines for their assembly. Jonathan Wells gave the update by saying, “If even one of these parts is missing or is placed in the wrong place, your engine will not run. So this device for assembling the flagellar motor is itself irreducibly complex. In fact, what we have here is irreducible complexity all the way down.

Image source: ATP Synthase: the cell’s power plant, via Discovery Institute.

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