Prokaryota: Machina ex Deus

Ó 2004 Art Chadwick and Helen Setterfield

[N.B. This paper is a work in progress]

What is life?

In spite of a century of progress in cellular and molecular biology, we still do not have a definitive answer. The best we can do for now is to describe some of the characteristics of all living organisms, and use these descriptions as a kind of filter to distinguish living from non-living. For example, viruses are complex biochemicals that are not considered to be alive by most biologists. Consider viral properties - they cannot reproduce by themselves, they do not exhibit responses to stimuli, they do not grow, etc.- and they are not classified among living organisms. They are treated as biochemicals - clever biochemicals, but biochemicals, nonetheless. Still we do not know what life is. But this inability to define life may soon change.

One of the most ambitious attempts to define life is being carried out by a group of scientists and bioengineers at a privately funded biotechnology firm in Maryland. These individuals have developed what they believe to be a protocol for creating life in a test tube. They are working with the simplest organism known, the tiny bacterium Mycoplasma genitalium, whose complete DNA sequence, including about 480 genes, is known. This organism lacks many of the genes required by other organisms because it lives as a parasite in the reproductive tissues of primates, and obtains many required materials from the host tissues. The investigators have been "knocking out" genes from its chromosome one at a time to determine which genes are essential to the organism, and which can be substituted by the culture medium, or are not essential. They anticipate that a lower limit will be reached at somewhere around 350 genes, beyond which the organism will not be viable with one less gene. They are then proposing to construct the organism from non-living components, to make a viable cell. If they succeed, we may someday know more about what it means to be "alive". Whatever the outcome, it will doubtless have significant impact on our understanding of the ultimate origin of life. We may learn that humans are clever enough to backtrack an existing living organism to its basic components, and with the expenditure of billions of dollars and many thousands of man-hours on the project, are able to recreate what already existed. In any case, it will reinforce the conviction that life could never have happened by accident in nature.

Another approach to learning about life was launched in 2001. A massive five-year project sponsored by International Business Machines, with an anticipated expenditure of $100 million was targeted to build a new generation of supercomputer with a million processors. The machine, named "Blue Gene" is expected to be the fastest supercomputer in the world. Its purpose? To be able to predict the three-dimensional shape of a small protein, given the amino acid sequence. The deciphering of the structure of one protein is expected to require a year of dedicated computational time even on this massive supercomputer. Why is it important to know the ultimate shape of a protein from its amino acid sequence? Because this would allow us the possibility of designing new proteins that would have predictable shapes and might carry out predictable functions. In light of the fact that such monstrous power is required to decipher the three-dimensional structure of a single protein, how can any thoughtful person conclude that such a molecule could achieve the right shape for a function essential for a living cell by chance? In its own way, IBM is furthering the conviction that life would require a Designer far more intelligent than ourselves.

Origin of the First Cell

Whether life itself could have originated without a Creator is an issue we have dealt with elsewhere on this site. It seem clear that with our present understanding of the laws of physics, chemistry and biology, life could not have arisen on this planet under any conceivable conditions. But undeniably, life is here. Where did life originate? Carbonaceous films thought to represent the remains of living organisms are found in the earliest sedimentary rocks on the earth's surface. Many evolutionists believe the first cells were formed on the earth by natural processes prior to about 3.5 billion years ago. These early carbonaceous films are ascribed to forms reminiscent of modern prokaryotic organisms, and are generally assigned to modern groups of these organisms. The Prokaryota, single-celled organisms without nuclei or subcellular organelles, include the many forms of bacteria and blue-green algae, as well as other life forms. Because they superficially appear to be simpler than eukaryotic organisms, prokaryotic cells have been considered precursors to eukaryotes, including ourselves, by many evolutionary scientists.

Considering the bacterial cell to be typical of the Prokaryota, let us examine some of the features of bacterial cells in an attempt to understand the amount of information required to specify a prokaryotic organism. Consider the two competing theories for origin of living cells: they were created by an intelligent Creator, or they resulted from the actions of mindless chance and natural processes. Distinguishing these two possible mechanisms will require that we look at some of the features of bacterial cells that might enable us to make an intelligent choice of whether chance or design more plausibly explains their existence.

Cell Division

As important as the origination of the first living cell may be, that life is certainly limited if the cell cannot reproduce itself. This is particularly a problem for the evolutionary hypothesis, since evolution requires reproductive processes to operate before it can manifest its posited ability to improve and refine living systems. It is essential that we look for evidences in nature that will tell us about the history of life. Let us begin by considering the mechanisms of cell division in modern bacteria. This will give us useful information for answering the following questions. On a prebiotic earth, 1) in the absence of cell division, what evolutionary pressures could be operative to induce the first cell to increase its complexity?

and,

2) regardless of the mechanisms by which division may have occurred in the primitive cell, could a dividing primitive cell have evolved the mechanisms utilized in modern cell divisional processes?

 We will consider these topics in an overlapping fashion, looking first at what cell division entails and working from there to the complexities of modern cellular division.

What is Required for Cell Division? A cell, any cell before it can divide in a fashion that will increase the meaningfulness of the system, must replicate its contents. The central core memory in the form of DNA (or RNA if such a system is considered possible) must be replicated so that two more or less equivalent copies exist in the cell. These two copies must then be separated from one another in such a way that they can segregate independently to the resultant daughter cells. The parent cell must also make copies of all other critical molecules of which it is composed; otherwise, the cell contents will become diluted by division. These criteria apply regardless of the nature and origin of the systems.

Replication: DNA to DNA The prokaryote, typified by the colon bacterium, E. coli, has no nucleus, and the cytoplasm does not contain organelles. The bacterial cell itself may be only 1-2 microns in diameter. The DNA molecule it contains is huge by comparison, about 1.4 mm in length, containing 4,639,221 bases. The DNA is thus 1000 times the length of the cell; its hydrated volume (volume when exposed to water outside the confines of the cell) exceeds that of the cell 1000 fold.

A bacterial cell replicates in 45 minutes. During that time it must not only duplicate proteins and other cellular constituents, and replicate the genetic material, but separate the two DNA strands so that one remains with each half of the dividing cell. DNA molecules in bacterial cells occur as intact circles. The processes involved in this division in modern bacterial cells are very sophisticated. We will consider most of the process in just the barest details.

In the cytoplasm, the double-stranded DNA circle is associated with specific positively charged proteins that shield the negative charges of the DNA. The molecule is condensed considerably by associating with specialized bacterial proteins called H-NS. These proteins, about 20,000 per cell, shrink the DNA loop down to a small fraction of its unbound length. Then the entire circle is supercoiled, much as the rubber band on a child’s model airplane propeller supercoils when it is overwound. Specialized proteins in the cell membrane attach to the daughter chromosomes, assuring that one copy ends up in each of the resultant cells. DNA replication itself requires nearly all of the 45 minute interval for completion. The synthesis of the new DNA strand is the work of an enzyme complex called DNA Polymerase. The system works as follows.

1) An initiation site (oriC) on the chromosome consisting of a series of 9 base and 13 base repeats rich in A-T pairs (melts easily) is recognized and associated with about 10-20 units of an initiation protein called DnaA. This binding results in the formation of a localized left-handed supercoil, which opens up the strands.

2) A second protein, DnaC recognizes the strands opened up by the DnaA complex and delivers a third protein, DnaB to the site.

3) The DnaB protein, a hexamer of six identical subunits, is a helicase, an enzyme specialized in unwinding the DNA helix so that the strands can be exposed and copied by the DNA Polymerase complex. The molecule forms a clamp , completely encircling each strand of DNA, opening up the helix as the copying process progresses along the molecule.

4) A fourth molecule, Ssb (Single strand binding) protein, binds to both strands in many copies, coating the exposed strands and preventing them from rejoining as they are opened by the helicase.

5) The polymerase is incapable of initiating DNA synthesis without being "primed" with a short stretch of RNA bases. A fifth protein called primase or RNA polymerase primase, binds to the helicase protein at the oriC site, forming a unit of primasome on each strand. The primase lays down a stretch of RNA primer complementary to the DNA strand , then drops off.

 The two strands of the DNA molecule run in opposite directions. Each strand is synthesized beginning with a ribose with its 5' hydroxide free and ending with a ribose with its 3' hydroxide free.The directionality of each strand is determined by which hydroxide group on the ribose sugar at an end of the strand is unlinked. Since the complementary strands of each DNA molecule run in opposite directions, one is referred to as the 5' - 3' strand and the complementary strand as 3' - 5'.

DNA Polymerase III, one of three DNA Polymerases in E. coli, and the enzyme responsible for replication, is only capable of reading and copying in one direction, 5'-3'. This enzyme is a sophisticated protein complex consisting of 10 different polypeptides, and is a dimer in its active form. The various subunits function either to anchor the enzyme to the DNA strand (beta subunits), to replicate the strand, or to keep the enzyme from falling off the strand prematurely.

6) DNA Polymerase III now copies the 5'-3' (leading) strand in the forward direction, and loops back the 3'-5' strand (lagging) so it can at the same time copy it in the 5'-3' direction, but in smaller increments, each of which must first be primed by the primase. The small pieces of RNA-primed DNA that result on the lagging strand are termed Okazaki fragments.

7) The RNA fragments produced on the lagging strand are removed by another polymerase, DNA Polymerase I. This enzyme simultaneously removes the RNA primer fragments, and replaces them with DNA.

8) DNA ligase next joins up the broken ends of the fragments to establish the integrity of the lagging strand.

All of these functions work in a highly coordinated manner to enable the complex to move along the strand copying both strands simultaneously at the rate of 500-1000 bases per second! 9) At specific termination sites in the DNA, a protein called Tus is bound which prevents the advance of the helicase, causing the Polymerase complex to fall off, ending replication. While this ends the replication phase, it does not finish the business of duplication. We are left with two interlocking circles of DNA that must now be separated. 10) A remarkable protein complex called either topoisomerase II, or DNA gyrase comes in to finish the work. This protein attaches itself to the cell membrane or some other substrate, and grabs the catenated circles. It cuts one of the molecules completely through, holds on to it, then grabs the other molecule and drags it through the break. The cut ends are then rejoined and the two new strands are ready for cell division to begin. It is probable that some molecule like topoisomerase holds on to the completed chromosome while being itself attached to the cell membrane, thus assuring as the cell dividsion completes, each daughter will have its own chromosome.

 What we have described so far is only the process of DNA replication, in the simplest kinds of cells, prokaryotes, where no nucleus is involved, where there is only one chromosome to worry about, and where only the simplest kinds of processes are carried out. I would not argue that information replication could not be done in a less efficient manner in a primitive cell. I would be tempted to ask what pathway one might take to get from the primitive cell to the condition of complexity represented in E.coli. Bacterial cells are among the earliest reported fossils. One would presume that bacterial cells in the earliest Precambrian faced the same kinds of problems as modern bacterial cells, and that they had therefore similar mechanisms for coping. But where did the information come from for developing the cellular complexity of even the simplest bacterium? All of the history of this earth is not sufficient time to generate even a single molecule with the information content of a single protein. The enzymes we are discussing in cellular processes are so complex one is tempted to anthropomorphize them! And we have just considered DNA replication!

The ATP Synthase Complex

All cells require ATP, Adenosine Triphosphate as an energy source in many biochemical processes.  ATP is formed from ADP (Adenosine Diphosphate) and inorganic phosphate, using energy derived from proton gradients across membranes.  Without ATP many critical cellular reactions, including the synthesis of DNA and RNA and proteins cannot take place.  Many of the common metabolic pathways are designed to produce ATP, the cellular fuel. These pathways and the multitude of enzymes they contain are themselves exceedingly complex and worthy of study in their own right. First let us take a hard look at the complex of proteins responsible for the manufacture of ATP.

ATP is formed in two ways in the cell. The most basic process occurs through substrate-level phosphorylation.In this reaction, energy is released and directly coupled with a second >reaction in which a molecule of ADP (ATP minus the terminal phosphate group) is joined with a molecule of phosphate . This kind of reaction occuring during the harvesting of energy from glucose by the cell, is responsible for all of the ATP available during respiration in the absence of a suitable electon receptor (usually oxygen). The amount of energy available from this process alone is so small that it can contribute but little to the maintenance of an organism. Bacteria, and all other living organisms make most of their ATP using another mechanism, the proton motive force.  Hydrogen ions are pumped outward through a membrane (the inner membrane of mitochondria or the cell membrane of bacteria) in the process of, and utilizing the energy from, the breakdown of sugar. This proton gradient represents a source of energy that can be tapped to promote the synthesis of ATP from ADP and phosphate ions. The membrane protein system responsible for this process is called the ATP Synthase Complex. In eukaryotic organisms, this process occurs in the mitochondrial membranes. Bacteria have no mitochondria, but the same complex of proteins found in eukaryotic mitochondria is present in the cell membrane of bacteria. ATP Synthase is composed of two separate complexes of proteins.  The first of these, the F0 complex resides in the membrane itself ). This complex consists of three different types of proteins, and is responsible for providing a channel for the flow of electrons through the membrane, and for converting the electrical energy into mechanical energy.  The second complex, called the F1 Complex, is in the cytoplasm of the bacterial cell (or matrix of the mitochondrion) and is responsible for converting the mechanical energy into chemical bond energy in ATP. 

The completely assembled ATP Synthase complex functions in much the same manner as an electric motor.  Clever experiments have elucidated the role of the F1 complex.  Protons flow from outside the membrane through a channel between the "a" (green) and "c" (yellow) subunits of the F0 complex. This flow results in rotation of the "c" rotor complex. Apparently, the passage of one proton moves one subunit past the proton channel, positioning the subsequent "c" protein adjacent to the channel. Thus, about four protons are necessary to rotate the "c" rotor through 120 degrees, the movement required to generate one molecule of ATP. The g protein (the shaft), anchored to the "c" complex by the e protein, is in turn rotated, causing the allosteric modification of the b subunits of the hexamer, and producing ATP. The process can be visualized as a rotating electrical motor. The ATP Synthase, invented prior to any man-made electrical motor, nonetheless contains all of the parts and functions in an exactly analogous manner. Electric motors can be converted into generators by supplying a source of external power. The experiment described above illustrates that the ATP Synthase Complex can be similarly affected by supplying ATP externally.

 

 

 

 

 

"Nature leads the way when it comes to motors on the molecular scale."

 

"If you could build a motor one millionth of a millimetre across, you could

fit a billion billion of them on a teaspoon. It seems incredible, but

biological systems already use molecular motors on this scale."

 

" . . . the rotary motor can be controlled by oxidation or reduction at the

metal centre, producing a device reminiscent of transmission systems in much

larger motors."

 

 

            "Perhaps in no other area of modern biology is the challenge posed

            by the extreme complexity and ingenuity of biological adaptations

            more apparent than in the fascinating new molecular world of the

            cell. Viewed down a light microscope at a magnification of some

            several hundred times, such as would have been possible in

            Darwin's time, a living cell is a relatively disappointing spectacle

            appearing only as an ever-changing and apparently disordered

            pattern of blobs and particles which, under the influence of unseen

            turbulent forces, are continually tossed haphazardly in all directions.

            To grasp the reality of life as it has been revealed by molecular

            biology, we must magnify a cell a thousand million times until it is

            twenty kilometres in diameter and resembles a giant airship large

            enough to cover a great city like London or New York. What we

            would then see would be an object of unparalleled complexity and

            adaptive design. On the surface of the cell we would see millions of

            openings, like the port holes of a vast space ship, opening and

            closing to allow a continual stream of materials to flow in and out. 

            If we were to enter one of these openings we would find ourselves

            in a world of supreme technology and bewildering complexity. We

            would see endless highly organized corridors and conduits

            branching in every direction away from the perimeter of the cell,

            some leading to the central memory bank in the nucleus and others

            to assembly plants and processing units. The nucleus itself would be

            a vast spherical chamber more than a kilometre in diameter,

            resembling a geodesic dome inside of which we would see, all

            neatly stacked together in ordered arrays, the miles of coiled chains

            of the DNA molecules. A huge range of products and raw materials

            would shuttle along all the manifold conduits in a highly ordered

            fashion to and from all the various assembly plants in the outer

            regions of the cell. 

 

            We would wonder at the level of control implicit in the movement

            of so many objects down so many seemingly endless conduits, all in

            perfect unison. We would see all around us, in every direction we

            looked, all sorts of robot-like machines. We would notice that the

            simplest of the functional components of the cell, the protein

            molecules, were astonishingly, complex pieces of molecular

            machinery, each one consisting of about three thousand atoms

            arranged in highly organized 3-D spatial conformation. We would

            wonder even more as we watched the strangely purposeful

            activities of these weird molecular machines, particularly when we

            realized that, despite all our accumulated knowledge of physics and

            chemistry, the task of designing one such molecular machine - that

            is one single functional protein molecule - would be completely

            beyond our capacity at present and will probably not be achieved

            until at least the beginning of the next century. Yet the life of the

            cell depends on the integrated activities of thousands, certainly tens,

            and probably hundreds of thousands of different protein molecules. 

 

            We would see that nearly every feature of our own advanced

            machines had its analogue in the cell: artificial languages and their

            decoding systems, memory banks for information storage and

            retrieval, elegant control systems regulating the automated

            assembly of parts and components, error fail-safe and proof-reading

            devices utilized for quality control, assembly processes involving

            the principle of prefabrication and modular construction. In fact, so

            deep would be the feeling of deja-vu, so persuasive the analogy,

            that much of the terminology we would use to describe this

            fascinating molecular reality would be borrowed from the world of

            late twentieth-century technology. 

 

            What we would be witnessing would be an object resembling an

            immense automated factory, a factory larger than a city and

            carrying out almost as many unique functions as all the

            manufacturing activities of man on earth. However, it would be a

            factory which would have one capacity not equalled in any of our

            own most advanced machines, for it would be capable of replicating

            its entire structure within a matter of a few hours. To witness such

            an act at a magnification of one thousand million times would be an

            awe-inspiring spectacle."

 

`

Next to mycoplasmas, E. coli is one of the most thoroughly & intensely studied

microorganisms (BTW, in 1979 T.D. Brock said

mycoplasmas were "of special evolutionary interest because of their

extremely simple cell structure" but in 1996 Dybvig & Voelker stated,

"Mycoplasmas can no longer be thought of as a simple organism."  Both

scientists used the word simple. Notice also, that with more time &

research, the 'simple' turned out to be awfully complex!.

 

"Even though the entire sequence

of the E. coli K-12 chromosomal DNA has been known for more than2 yrs., we are still

far from knowing all of the details of how the cell operates, lives,

replicates, coordinates and adapts to changing circumstances . . . the

number of experimental journal articles on aspects of the basic biology of

E. coli has increased from an average of 78 per month in 1996 to an average of

94 per month. today . . . new biological information about this well-studied

organism continues to roll in. New metabolic capabilities are discovered and

are connected to underlying genes. There are new regulation systems, new

transport systems, and more information on cellular constituents & cellular

processes" (p. 342).

 

" . . . but how many regulators are needed to maintain coordination of

expression of the genes and correct interaction among the gene products?

Regulation systems are not the same in all bacteria, and we still do not

have all of the information for the regulatory networks of even 1 bacterial

species . . . the minmal set of genes and proteins necessary for life of an

independently replicating cell does not have an easy answer" (p. 375)

 

"Experimentation into details of the biology of E. coli continues unabated

today, and the numbers of papers published annually continues to increase .

. . . not all enzymes & pathways in E. coli are known . . . besides genes

for unknown enzymes, we have data for enzymes that don't have genes. There

are 55 enzymes of E. coli that have been isolated, purified and

characterized over the years, but their genes have never been identified"

(p. 361).

 

Finally, "The advent of massive DNA-sequencing technology and the completion

to date of more than twenty microbial genomes that are now available to the public have

not brought us (yet) to a complete understanding of exactly how a single

free-living cell functions and adapts to changing environments" (p. 380).

 

           

 

Denton M.J., "Evolution: A Theory in Crisis," Burnett Books: London, 1985, pp.328-329)

 

 

FERINGA, B. L. Nanotechnology: In control of molecular motion. NATURE, 9 Nov.2000, Volume 408 No. 6809, p. 151

Riley & Serres. 2000. Interim report on genomics of Escherichia coli" Annual Review of Microbiology,'. p. 341ff

 

[End of TEXT]

 

 

 

 

Science has a review article in the issue for March 8, 2002, on the complex machinery (the authors' word) needed to ensure that newly synthesized proteins fold properly. The abstract is given below along with the first paragraph, which states the problem being addressed. A misfolded protein probably will not carry out its function, and may prevent the proper function of other molecules in the cell. The need for chaperones adds to the complexity of protein production in cells. Molecular Chaperones in the Cytosol: From Nascent Chain to Folded Protein F. Ulrich Hartl* and Manajit Hayer-Hartl Efficient folding of many newly synthesized proteins depends on assistance from molecular chaperones, which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Nascent chain-binding chaperones, including trigger factor, Hsp70, and prefoldin, stabilize elongating chains on ribosomes in a nonaggregated state. Folding in the cytosol is achieved either on controlled chain release from these factors or after transfer of newly synthesized proteins to downstream chaperones, such as the chaperonins. These are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. Understanding how the thousands of different proteins synthesized in a cell use this chaperone machinery has profound implications for biotechnology and medicine. Department of Cellular Biochemistry, Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany. *To whom correspondence should be addressed. E-mail: uhartl@biochem.mpg.de To become functionally active, newly synthesized protein chains must fold to unique three-dimensional structures. How this is accomplished remains a fundamental problem in biology. Although it is firmly established from refolding experiments in vitro that the native fold of a protein is encoded in its amino acid sequence (1), protein folding inside cells is not generally a spontaneous process. Evidence accumulated over the last decade indicates that many newly synthesized proteins require a complex cellular machinery of molecular chaperones and the input of metabolic energy to reach their native states efficiently (2-5). The various chaperone factors protect nonnative protein chains from misfolding and aggregation, but do not contribute conformational information to the folding process. Here we focus on recent advances in our mechanistic understanding of de novo protein folding in the cytosol and seek to provide a coherent view of the overall flux of newly synthesized proteins through the chaperone system. http://www.sciencemag.org/cgi/content/full/295/5561/1852

 

." Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6854– 6859, June 1998

> The Bacterial Flagellar Cap as the Rotary Promoter of Flagellin Self-Assembly. Koji Yonekura, Saori Maki, David Gene Morgan, David J. DeRosier, Ferenc Vonderviszt, Katsumi Imada, and Keiichi Namba. Science, 290, Dec 15 2000: 2148-2152. The growth of the bacterial flagellar filament occurs at its distal end by self-assembly of flagellin transported from the cytoplasm through the narrow central channel. The cap at the growing end is essential for its growth, remaining stably attached while permitting the flagellin insertion. In order to understand the assembly mechanism, we used electron microscopy to study the structures of the cap-filament complex and isolated cap dimer. Five leg-like anchor domains of the pentameric cap flexibly adjusted their conformations to keep just one flagellin binding site open, indicating a cap rotation mechanism to promote the flagellin self-assembly. This represents one of the most dynamic movements in protein structures. 07 September 2000 Nature 407, 41 - 47 (2000) c Macmillan Publishers Ltd. Microtubule motors in mitosis DAVID J. SHARP, GREGORY C. ROGERS & JONATHAN M. SCHOLEY The mitotic spindle uses microtubule-based motor proteins to assemble itself and to segregate sister chromatids. It is becoming clear that motors invoke several distinct mechanisms to generate the forces that drive mitosis. Moreover, in carrying out its function, the spindle appears to pass through a series of transient steady-state structures, each established by a delicate balance of forces generated by multiple complementary and antagonistic motors. Transitions from one steady state to the next can occur when a change in the activity of a subset of mitotic motors tips the balance. Sharp, D. J. et al. Microtubule motors in mitosis Nature 407, 41-47 (2000) Review Article |Summary|Full text|PDF(4149K) 407041ai1.avi AVI movie file (740K) 407041ai2.doc Microsoft Word document (20K) Movie legend