Basic Functions Within a Living Cell

Introduction

…the fact that our knowledge is very incomplete; thus a great deal remains to be discovered about how DNA provides the instructions to build living things.

Alberts et al., Essential Cell Biology

As we have seen in previous chapters, major mysteries remain concerning how life originated and how complex biological systems evolved. But the source of those mysteries don’t lie exclusively in the past: the same magnitude of mysteries manifest in every living cell. In some manner, the solution to those mysteries will be discovered within the molecular world of living cells, or as I suggest, within the Vicarian Domain.

In contrast to the current state of biology and bio-chemistry, I can explain every system in a pre-1970 automobile, how everything inter-relates, how the thing moves from one place to another. With a bit of research, I could expound upon the origin of the automobile, and its current use, from an economic, cultural, historical or anthropological perspective. When a car quits running, there is always a reason, one that can generally be fixed. No mysteries, in other words. The complete and unbroken causal links from pushing down on the throttle to increase the gas/air mixture drawn from the carburetor through the intake manifold and into the firing chamber; intake valve closing (driven by push rods and the cam shaft) and the piston compressing the mixture until the spark plug ignites the mixture in a small explosion (the spark plug connected to wires emanating from the distributor that send the charge when the rotor touches that wire) driving the piston down, contributing to the circular motion of the crankshaft; the crankshaft with one side of a pressure plate of the clutch that drives the transmission when the plates contact each other, providing the circular motion to the drive shaft and into the differential, turning the motion laterally with a fixed gear ratio to the axle that spins the wheel. And the car moves….

Can the same thing be said about a living cell? Do scientists understand all of the processes and how they relate? For instance, what triggers the creation of energy within the mitochondria, and how does the cell ‘know’ how and when to engage those bio-chemical reactions? Within the three dimensional space of a living cell, how do the necessary molecules (proteins, amino acids, carbon, sugars, etc.) physically get from where they are to where they are needed? What controls all of these highly complex processes and reactions? At the macro level, we understand how blood flows and nerves interact with muscle tissue, but do we possess the same level of understanding at the cellular level?

If the explanation is purely bio-chemical, why can’t scientists simply place the right combination of molecules in the correct portions and configurations to replicate the life processes within a cell?

Do scientists understand how any part of a genotype translates the coded DNA into an actual organ, limb or instinct? In even one case?

As we will review shortly, the answer to all of these questions, and many more like them, is an emphatic “no”. From Essential Cell Biology (Fifth Edition, 2019), the text that will serve as the central source for this chapter, poses several major unanswered questions:

· How did cells arise on Earth?

· How did they evolve in so many ways?

· How is it possible for billions of cells to seamlessly cooperate?

Before delving into the detailed functions within a living cell, consider the following from iGenetics:

After mRNAs are generated by RNA processing, they are exported from the nucleus through the nuclear pore complex to the cytoplasm. The mRNA is exported in a complex with a number of proteins. Some of those proteins are recruited to an mRNA during the transcription and splicing processes, illustrating the tight linkage between transcription, splicing, and nuclear export. The mRNA export process is perhaps the most intricate of the export systems using the nuclear pore complexes, involving many quality control steps. Once in the cytoplasm, the mRNA may be translated immediately, or stored for later translation.

Peter Russell, iGenetics

“Generated”, “exported”, “recruited”, “tight linkage”, “quality control”, “translated” or “stored for later”. I realize that scientists must use metaphorical language to describe such processes, but even so, something drives these molecules to behave in a very specific manner. Another example from iGenetics:

After determination, the most spectacular aspect of development takes place: differentiation. Differentiation is the process by which determined cells undergo cell-specific identities, such as nerve cells, skin cells….and so on in animals…Differentiation in most cases results from differential gene expression, rather than from a differential loss of DNA that leaves different sets of genes in different cell types. That is, expression of different sets of genes in different kinds of determined cells leads to different proteins in the cells, and the proteins guide the progression to the various differentiated states.

Peter Russell, iGenetics

I suspect that most scientists expect to better understand how these processes take place within every living cell by further expansion of bio-chemical discoveries. Some description of valences and attractions/repulsions, folding and fitting, and so on. But I believe that such further discoveries, ones that proceed along existing paths, will ultimately prove inadequate. The next quote from Richard Lewontin in his Triple Helix poses the relevant comparison:

The messenger RNA molecule that is the immediate copy of a gene that is being read by the cell must move out of the nucleus and into the cytoplasm in order to take part in the synthesis of proteins. In the cytoplasm it must be inserted into a ribosome, the machine that actually manufactures a protein according to the specification carried by the RNA. This process and all others like it in the cell take time and occupy space and are quite unlike the picture of what happens when billions of small molecules interact with each other by bouncing around in a solution.

Richard Lewontin, Triple Helix

And that’s the question: what is the difference between the molecular behavior in a living cell, and the non-behavior in a solution of the same molecules just swished around together? Why can’t the processes within a living cell be duplicated by science from some assembly of the basic parts? Sure, scientists use existing living cells to support cloning and creating strands of DNA that the cells wouldn’t otherwise manufacture, but why not put the basic ingredients together and spark similar bio-chemical processes? Why can’t science create life from non-life, using the same ingredients?

Cells deploy a wide variety of mechanisms to make sure that each of their chemical reactions occurs at the proper rate, time and place. [p. 39]

This is the locus of the mystery. How, within a cell, do all the transactions take place when, where and how they are necessary?

We assume that biological beings emerged from the physical/chemical substrate, and are subject to physical laws and limitations, including cause and effect.

At the macro level we can trace back antecedent conditions that led to a particular event. For example, take the major eruption on Mount St Helens on May 18, 1980. It didn’t just happen. There exists a thorough and unmysterious explanation for the eruption. Even if humans don’t possess all the relevant geological facts.

In the human world, every machine requires an operator, a driver, or a programmer. For molecular machines, many of which are far more complex than many human machines, how are they operated and/or programmed?

We understand many of the bio-chemical processes in the same way we understand how an internal combustion engine works. And we also understand many of the intra-cellular signals that trigger a particular event. But what signals the trigger? How and why? If you take any particular event, say the translation by a ribosome of a protein, how did that particular action by a particular ribosome to manufacture a particular protein begin? We know DNA has the gene for that protein, and we know that the mRNA transcribes that gene from the DNA strand, but how does anything in the cell know that that particular protein is required, when and where? And this question stands for the tens of thousands of millions of actions that occur constantly in every cell in every living thing:

Each cell can be viewed as a tiny chemical factory, performing many millions of reactions every second. [p. 81]

And just because atoms and molecules can combine in a particular way, why do they do so in a particular way at a particular location and time?

Once we descend into the actual functions of various macromolecules, the questions will become more specific, and hopefully more suggestive.

Overview

The first thing we need to point out is how difficult it is to determine exactly what takes place within a living cell. Most of the internal structures are transparent and colorless. [p. 8] It’s almost impossible to view the molecular processes as they occur. [p. 10] Large macromolecules can be viewed in crystalline stasis, but individual atoms remain invisible. Structures smaller than .02 mm – about half the wavelength of visible light cannot normally be resolved. [p. 9] In sum, it is currently impossible to view, map, or determine the behavior of proteins. Sample proteins must be processed, or frozen, or crystalized before their atomic structure and shape can be determined.

Given these visible restrictions, it’s remarkable how much science has been able to determine what takes place within a cell. Even so, this limitation will figure prominently in the argument for the viable, meaningful and unknown activities that must take place within the Vicarian Domain.

An interesting, and necessary, property of the cell is its ability to remain chemically neutral by the presence of buffers: mixtures of weak acids and bases that will adjust proton concentration around PH7 by releasing protons (acids) or taking them up (bases) whenever the PH changes. This keeps the PH of a cell relatively constant under a variety of conditions. [p. 50]

Although about five hundred amino acids are known to chemistry, only twenty of those serve as the fundamental components of life, from which virtually all proteins are assembled.

David Quammen, The Tangled Tree

One of the mysteries (among so many) is how the precise set of 20 amino acids came to be chosen. There is no obvious chemical reason why other amino acids could not have served just as well. But once selected it could not be changed, as too much chemistry had evolved to exploit it. Switching amino acids would require an organism to retool its entire metabolism to cope with the new building blocks. [p. 56]

Proteins, which consist half the dry mass of the cell, lie at the center of life’s chemistry. [p. 56]

A related mystery is that only L-form amino acids are found in proteins. [p. 56] Amino acids exist in two forms – D (right handed) and L (left handed). But every living thing contains L-amino acids. This is known as homochirality. Life could exist with either one or the other, but the two would be chemically incompatible in the same way a left handed glove won’t fit properly on your right. Once the original decision was made to go with L-amino acids, every subsequent organic entity utilized the one form of amino acid to construct proteins.

One of the questions I will repeatedly pose as we explore specific molecular activities is how the molecules, particularly the small ones, get from one place to another within the cell, and arrive at the precise location, in the precise orientation, at the right time. The text puts the question this way:

How do enzymes and their substrates, which are present in relatively small amounts in the cytosol of a cell, manage to find each other. And how do they do it so quickly? [p. 99]

Science answers as follows:

A typical enzyme can capture and process about a thousand substrate molecules every second.

Rapid binding is possible because molecular motions are enormously fast – because of heat energy, molecules are in constant motion and consequently will explore the cytosolic space very efficiently by wandering randomly through it – a process call diffusion. In this way, every molecule in the cytosol collides with a huge number of other molecules each second. As these molecules in solution collide and bounce off one another, an individual molecule moves first one way and then another, its path constituting a random walk. [p. 99]

Once again science resorts to a random process. And yet this can’t be right. There must be another explanation. For one thing...

A living cell contains thousands of different enzymes, many of which are operating at the same time in the same small volume of the cytosol. By their catalytic action, enzymes generate a complex web of metabolic pathways, each composed of chains of chemical reaction in which the product of one enzyme becomes the substrate of the next. In this maze of pathways, there are many branch points where different enzymes compete for the same substrate. The system is so complex that elaborate controls are required to regulate when and how rapidly each reaction occurs. [my emphasis] [p. 150]

For these enzymes to operate randomly in such a complex way that elaborate control systems are required is impossible. We will see so many instances where specific small molecules are required to complete a chain or provide additional coding, where a particular molecule must be ‘recruited’. Besides,

at any one time, a typical eukaryotic cell carries out thousands of chemical reactions, many of which are mutually incompatible. [p. 495]

This means that some form of command and control is necessary in order to segregate and time the reactions in a way beneficial to the cell. Further evidence for such control happens when the molecules are removed from the context of a living cell:

If the cells of an organ such as the liver are broken apart and their contents mixed together in a test tube, the result is chemical chaos. [p. 495]

To think that a random jumble of small molecules and macromolecules existing and vibrating within the same cytosol could come together in just the right way at just the right time seriously stretches one’s credulity.

Non-covalent bonds can also stabilize associations between any two macro-molecules, as long as their surfaces closely match. Such associations allow macromolecules to be used as building blocks for the formation of much larger structures. For example, proteins often bind together into multi-protein complexes that function as intricate machines with multiple moving parts, carrying out such complex tasks as DNA replication and protein synthesis. In fact, noncovalent bonds account for a great deal of the complex chemistry that makes life possible. [p. 63]

And this takes place in three dimensions. In other words, in order for the non-covalent bonds to adhere between two molecules, the three-dimensional fit must be nearly perfect, and the molecules must approach very very close, as the range of non-covalent forces is very small.

If this solution was viable, it would be possible to design an experiment where small molecules were simulated by similarly shaped object, made out of very light material and embedded with magnets at specific points representing potential non-covalent bonds. Put thousands of these things in a container with a powerful fan blowing all the pieces into the air and jumbling together and see what you get. No doubt some of the pieces would join, but this wouldn’t prove anything because to simulate a cascade of reactions equivalent to a metabolic pathway, a specific series of joinings and unjoinings would be required. That being be case, it wouldn’t be possible to properly set up the experiment because it would be impossible to randomly introduce the control mechanisms required to manage the direction and a specific metabolic path.

How various molecules (mRNA, ribosomes, proteins, all macromolecules, etc.) get from one place to another will be a constant question as we examine specific cases. In only one case do we have a specific, mechanical answer, and that involves moving a sack of proteins from one place in the cell to another. And that example carries serious questions of its own.

Protein Synthesis

The following sections delve into several processes required to create and locate proteins within a living cell. As a non-scientist I will attempt to reflect my understanding of these processes in some detail. The purpose is to provide the necessary context to understand the principle claim within this site: that the key to every biological mystery we have encountered, including how life originated and how complex biological structures evolved, resides within the molecular world of a living cell, what I refer to as the Vicarian Domain.

This particular journey will begin with RNA Transcription, followed by RNA splicing, RNA export, RNA translation, protein synthesis and protein transport. This particular cycle, one of dozens that could have been selected as an example, exists in every living cell on Earth, and likely existed in the first cell almost 4 billion years ago.

Incidentally, this is the cycle that Dawkins suggested came into existence by pure chance:

Suppose we want to suggest, for instance, that life began when both DNA and its protein-based replication machinery spontaneously chanced to come into existence. We can allow ourselves the luxury of such an extravagant theory, provided that the odds against this coincidence occurring on a planet do not exceed 100 billion billion to one.

Richard Dawkins, The Blind Watchmaker

It will soon become evident how ridiculous such a suggestion is.

ATP

Every system has a natural tendency to become increasingly disordered. To halt this inevitable progression, every living thing must obtain energy, and constantly apply it. The primary source of energy within a cell is ATP.

We will encounter many instances where ATP is required to complete a particular biochemical reaction.

The Code

The ability to translate mRNAs accurately into proteins is a fundamental feature of all life on earth. [p. 255]

All cells, from bacteria to those in humans, express their genetic information in this way – a principle so fundamental that it has been termed the central dogma of molecular biology. [p. 228]

Before we delve into RNA transcription, a few words about what is about to be transcribed. The DNA molecule in its famous double helix consists of a long series of four nucleotides: adenine (A), cytosine (C), guanine (G), and thymine (T). As far as we know, this four-letter code determines everything that takes place within a living organism.

Regardless of how the various long non-coding RNAs operate – or what exactly each of them does – the discovery of this large class of RNAs reinforces the idea that a eukaryotic genome contains information that provides not only an inventory of the molecules and structures every cell must make, but also a set of instructions for how and when to assemble these parts to guide the growth and development of a complete organism. [p. 292]

There is no other known source for planning, architecture, coordination, communication, development or differentiation within a living cell. There is no brain or nervous system within a cell. No arms, legs, hands, fins or feet.

A large part of the human genome does not code for proteins or RNA, but serves a regulatory function that allows/promotes extensive complexity. [p. 35]

The specific sequence of these four letters provides the basis for specific genes within DNA. These genes are spread out among the 23 chromosomes within a human. Each DNA molecule can be up to 250 million nucleotides long, for a total of around 3 billion nucleotide pairs within the human genome. [p. 231].

Less than 2% of the human genome codes for proteins, [p. 322] with 4.5% of the genome highly conserved. About 10% of the human genome contains sequences that truly matter [p. 323] but we don’t know what it does.

The human genome has about 19,000 protein-coding genes. [p. 37] What follows is a description of how those genes begin the process of protein synthesis.

Each gene can be transcribed, and its RNA translated, at different times, providing the cell with a way to make vast quantities of some proteins and tiny quantities of others. Moreover…a cell can change (or regulate) the expression of each of its genes according to the needs of the moment. [p. 228]

While tactical explanations exist for several ways that gene expression is controlled, how the cell, in its entirety, ‘knows’ what genes and/or their product is required remains a telling mystery.

RNA Transcription

Below is a diagram of DNA. What is important to note is the relationship between the nucleotides (A, C, T, G) and the fact that C always bonds with G, and A always with T. This means that the two strands mirror each other, and one of them can be used as a template to recreate the other.

And that is what takes place with RNA transcription. An RNA molecule is assembled using a particular part of the DNA molecule, one that includes a specific gene for a specific protein so that the RNA molecule can carry this coded piece from the nucleus, where the DNA resides, into the cytosol, where the ribosomes wait for such a molecule to manufacture the needed protein.

How does this process begin? According to the text…

When an RNA polymerase collides randomly with a DNA molecule the enzyme sticks weakly to the double helix and then slides rapidly along its length. RNA polymerase latches on tightly only after it has encountered a gene region called a promoter, which contains a specific sequence of nucleotides that lies immediately upstream of the starting point for RNA synthesis. [p. 233]

The RNA polymerase is the factory that will produce the new RNA molecule. But let’s try and imagine what this ‘random’ process really means. When the DNA molecules within a cell’s nucleus are unraveled they become invisible to humans. We only see the distinctive chromosomes when they are bundled together as part of the process of mitosis – that is, in preparation for cell division.

But most of the time their stringy mass simply floats within the nucleus. As we noted above, each one of these DNA molecules might consist of as many as 250 million base pairs.

To put this in perspective, if each of the nucleotide base pairs was the size of a golf ball, the molecule would stretch over 6000 miles – roughly the distance between New York City and Tokyo. And that is just one of 23 DNA molecules within the nucleus. A typical gene consists of about 1500 base pairs, about 210 feet along that golf-ball size molecule that stretches for thousands of miles. That’s needle in the haystack territory.

And we know that the transcription of a particular gene is not random. While the cell has several ways to regulate gene expression, the most common is to regulate the initiation of transcription. [p. 259] Gene expression, and the creation of proteins, is tightly controlled within a cell. Gene expression is a complex process by which cells selectively direct the synthesis of the many thousands of proteins and RNAs encoded in their genome.

[p. 268]:

…most eukaryotic transcription regulators work as part of a large “committee” of regulatory proteins, all of which cooperate to express the gene in the right cell type, in response to the right conditions, at the right time, and in the required amount.

In addition to being able to switch individual genes on and off, all cells…need to coordinate the expression of different genes. When a eukaryotic cell receives a signal to proliferate…a number of hitherto unexpressed genes are turned on together to set in motion the events that lead eventually to cell division. [p. 279]

Of the approximately 19,000 protein-coding genes, only a subset of them are being produced at any particular time within a particular cell, perhaps 5000 to 15,000. [p. 270]

In order to control what proteins are created, and how many of them, something within the cell has to determine where the genes are located on the multitude of DNA molecules, and how to get the RNA polymerase in place to begin transcription. This is not a random process. There is some form of control and coordination taking place that we don’t yet understand. Even as we identify specific regulators (see figure 8-13) we don’t know how those regulators come to be placed. And this will be a common theme as we examine how these macromolecules behave. We continue to discover discrete mechanisms here and there that help explain how a particular set of biochemical reactions take place, but we have no understanding of how all the proper molecules are perfectly placed in perfect timing to contribute to the reaction. For example:

Regulatory DNA sequences [can be] very long (sometimes spanning more than 100,000 nucleotide pairs) and act as molecular microprocessors, integrating information from a variety of signals into a command that determines how often transcription of the gene is initiated. [p. 271]

Consider the figure directly below (8-13):

Note that it requires this molecular ‘mediator’ to bring together all of the components necessary to produce the proper RNA molecule, and therefore the proper protein. This is in essence a roving factory that connects various parts of a DNA molecule in order to produce the proper RNA molecule and in the proper numbers. Every one of those colors in the figure above [figure 8-13] represents a different set of molecules that work in close coordination through the mediator. These macromolecules move, react, construct, read, and arrange various other molecules.

We take a closer look at the RNA polymerase below [figure 7-7]. The RNA polymerase (pale blue) moves along the DNA molecule and unravels it as it goes. Breaking the covalent bonds that keep the DNA helix together requires energy. In most biomolecular reactions, the source of energy is provided by ATP.

After the single-stranded RNA gets extruded out of one side, the two strands of DNA are reformed.

Does anything in this process appear random? For instance, the RNA polymerase requires a supply of four different ribonucleotides (small molecules) in order to assemble the new RNA strand, but it doesn’t ‘know’ which of them it requires next until it ‘reads’ the DNA strand.

In our physical and chemical universe, how do these small molecules appear at the right place at the right time? How are they physically and/or chemically manipulated in order to be properly selected and placed? In order for the needed reactions to take place, the two molecules (the growing RNA chain and the next nucleotide) need to be physically/chemically brought into close proximity and positioned properly in order for the correct bond to take place. The only explanation we currently possess for the movement of these small molecules is ‘random walking’, a supposedly random process.

RNA transcription is an insanely complex and ancient cellular process. As far as we know, this process using these specific molecules operate in every living cell, and likely the first cell that existed some 4 billion years ago.

RNA Splicing

To produce an mRNA in a eukaryotic cell, the entire length of the gene, introns as well as exons, is transcribed into RNA. [p. 239] This is interesting because the introns – various lengths of coding – are not required to produce the protein. In fact, if even one incorrectly positioned nucleotide is included in the spliced mRNA, the protein will not be assembled correctly.

Splicing often takes place as the mRNA is being transcripted, or shortly after. As can be seen below in the figure [7-22] the process is quite complex, requiring over 200 proteins to complete it.

The explanations for these processes are largely tactical and local, in that they don’t consider the larger, strategic picture. The immediate reactions are described and the molecules involved, without consideration for how they arrived at the location, why they are performing a particular function, or how the local actions contribute to the health and needs of the entire cell.

None of this is accidental or random. Known physical and chemical processes are inadequate to provide a wholistic explanation as to how and why these macromolecules behave as they do. We haven’t discerned any form of coordination, communication, planning or management within the cell, as if all these actions are just randomly taking place without any impetus other than the influence of local chemical and physical states. And these biological processes are effectively eternal, part of a living perpetual motion machine.

RNA Export

After the mRNA molecule is transcribed and spliced, it must be removed from the nucleus and transported through the nucleus wall and into the cytosol, where it will rendezvous with a ribosome.

Before we look at that process in some detail, let’s provide some physical context. The average size of the nucleus of a cell is about 6 micro meters (6 * 10-6). The length of a typical RNA molecule ranges between 1-2 nano meters (1 – 2 * 10-9). Changing the scale to something we can grasp, if an RNA molecule is 2 inches long, the nucleus of the cell would be 6000 inches in diameter, or 500 feet. Assuming for the sake of argument that the nucleus is spherical, the volume within the nucleus would be over 65 million cubic feet (Volume=4/3 * π * r3). So depending on where in the nucleus the RNA was created, this 2 inch piece of thread needs to travel anywhere from 20 to 200 feet (200 to 1200 times its length), to reach the boundary between the nucleus and the cytosol. Once there, it needs to find a port that will lead to the cytosol:

Peter Russell, iGenetics

As Russell indicates, the transport of mRNA from the nucleus to the cytosol is highly selective: only correctly processed mRNA are exported and therefore available to be translated. [p. 242] How is this accomplished? Every mRNA molecule potentially differs, so how does the screening take place? If even one coding element is transcripted or spliced incorrectly, the mRNA will produce the wrong protein. Somehow, of all the mRNA fragments floating around the nucleus, special proteins bind to a particular mRNA molecule, signaling that it is ready for export (see figure 7-25 below). Presumably these binding proteins are able to distinguish faulty mRNA from those that are viable.

Once the mRNA is properly marked and reaches a nuclear pore, it gets processed for export into the cytosol. (see figure 7-25 below)

A nuclear pore is a large, elaborate structure composed of a complex of about 30 different proteins. [p. 503] These proteins operate this molecular gate at an amazing speed, rapidly pumping macromolecules in both directions through each pore. [p. 504] The nuclear pore is a sophisticated molecular machine, one that carefully and skillfully attracts, screens, and processes molecules in and out of the nucleus. There is nothing accidental or random in this process. It is purposeful, deliberate and focused, elements that cannot reasonably be ascribed solely to physical and chemical properties.

Basic Functions Within a Living Cell

Introduction

Overview

Protein Synthesis

ATP

The Code

RNA Transcription

RNA Splicing

RNA Export

Please go to Living Cell (2) to continue.

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