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© Karl Simpson
 


DNA MAKES RNA MAKES PROTEIN

 

DIAGRAM SHOWING INFORMATION FLOW IN A EUKARYOTIC CELL.

At every stage, within the cell, feedback processes are in operation. Biochemistry is self regulating. Note that RNA, more than DNA appears to play a leading role in information flow. Proteins and the proteome have perhaps the most profound functional impact. The metabolome is more diffuse, yet also more linked with the environment, be it surrounding cells or the ecology of which the cell is a part. The outer cell membrane links both metabolome and proteome to the outside. See following text

 

 

The biotechnology and pharmaceutical sectors are currently very excited about the possibilities opened up by the new genomics, proteomics and - in the future - metabolomics. What are these terms and how do they fit into our understanding of biology? The following is a primer for those who might be interested. Nucleic acids such as DNA and RNA (genome and transcriptome) are responsible primarily for information flow. Proteins and metabolism (proteome and metabolome) are primarily responsible for providing the raw materials and energy to maintain information flow and assure reproduction.

The title of this piece forms the "Central Dogma" of molecular biology, which has not been found wanting in the past fifty years. It has, of course, been nuanced and annotated. Before life was cast in its present mould it seems likely that there was a pivotal short period during the "origin of life" on Earth. During this "origin" phase a number of biological algorithms were explored, before the robust system of today was "cast in stone". The precursors to today's genetic material may have been variants of the familiar pyridines and pyramidines bound to some suitable chemical substrate - another sugar-phosphate chemical backbone or potentially clay derived from volcanic rocks by erosion and sedimentation.

It is easy to overlook the fact that the manipulation of genetic material also demands energy transactions. Today energy-rich molecules are synthesised and the energy subsequently liberated through a maze of protein-catalysed oxidation-reduction chemical reactions. At the earliest stages of life we suspect that RNA played a key role in catalysis, effectively substituting for protein. That early function is maintained in the most fundamental of macromolecular complexes, the ribosome. The ribosome is effectively a complex of catalytic RNA molecules and modulating proteins. The ability of the ribosome to translate a specific nucleotide sequence into a corresponding amino-acid sequence is the cornerstone of life. Ribosomes "translate" an RNA sequence into protein. DNA is copied to RNA in a process known as "transcription".

DNA --> transcription --> RNA -->translation --> Protein

Transcription is carried out by a group of enzymes, the DNA dependent RNA polymerases.

Life "as we know it" is the process which perpetuates DNA through the medium of the living cell. At its simplest, the living cell consists of genetic material, DNA, a cell membrane which separates in from out and a protein catalysed chemical activity (metabolism) which through the medium of RNA assures regeneration of protein (and DNA and RNA). At the level of the ribosome, RNA continues to play a role in "metabolism" or cell chemistry. The recent discovery of "ribozymes", RNA-based enzymes, or metabolically active RNA molecules, shows that proteins do not have an exclusive license to manipulate cell chemistry. It is possible that other macromolecules such as lipids or sugars might also challenge protein's exclusivity.

The mammalian red blood cell, which cannot replicate, contains no DNA, but nonetheless survives for about 150 days on the basis of long-lived RNA "messengers". So life as an immediate ongoing process is the manifestation of the chemistry of living cells. Changes in cell chemistry can be brought about on many levels. On the simplest level variations in pH or the concentration of the products of a chemical reaction can affect the rate of a reaction. At the level of proteins, pH, or levels of chemicals can influence the catalytic function of a protein, accelerating or slowing the rate of a chemical reaction. Chemical reactions on a protein template occur at an "active site". The exact physical shape - and function - of the active site may be modulated by other sites sensitive to the components of a specific chemical reaction or to other modulating agents such as hormones or "co-enzymes". A metabolic pathway is then a chain of chemical reactions mediated by a succession of different enzymes. Metabolism is the sum total of cellular chemistry or organism chemistry.

Although metabolism owes much to catalytic proteins (enzymes), it is also influenced by other changes in the cellular environment, creating a vastly complicated network of interactions in both time and space. As a biochemist who began his university studies in 1970, I am inclined to go back to my roots and refer to the assemblage of metabolic processes as "biochemistry" or quite simply the chemistry of life. Today's buzzword for biochemistry is "metabolomics". It has an undeniable elegance in the lineage of genomics and proteomics.

Mankind loves hierarchies. Society tends to be organised hierarchically. So a company has a CEO. To him answer a number of Vice Presidents. A number of Directors report to the Vice Presidents and Managers report to the Directors. Foremen report to managers and workers execute tasks. Biology lulls us into complacency by demonstrating some hierarchical organisation like; DNA goes to RNA goes to Protein. But "it ain't that simple".

Life is a cyclic process in which all components, including non-organic components have vital roles to play. The environment and ecology have critical impacts on the future of a living cell in isolation, or as part of an organism. DNA, which is placed at the pinnacle of life's hierarchy has a fairly lowly corporate equivalent; "archivist" or keeper of records. It is difficult to pinpoint the CEO in living processes. The Gaia hypothesis of Lovelock et al tends to resolve the problem by saying that all life in our planetary ecosystem is one and that the planetary ecosystem as a whole is the CEO.

In Gaia, hierarchies do have a role to play for assuredly and demonstrably, DNA --> RNA --> Protein. Equally, the primary sequence of a protein adopts secondary structures such as alpha helix or beta sheet, which adopt the tertiary structures of a folded protein - such as myoglobin. Equally, several proteins can form quaternary structures or macromolecular complexes such as the four subunit haemoglobin molecule. Macromolecular complexes can also include nucleic acids and lipids in such structures as ribosomes or trans-membrane molecular complexes. However, despite the obvious economies of information flow that are obtained by assembling structures from simple components, life is complex. Cellular chemistry is highly complicated and responsive to the environment of the cell. A cell can be part of a multi-cellular organism or a free living bacterium or amoeba. The division of life into nucleated eukaryotic cells and bacteria is somewhat simplistic. Comfortable in our hierarchical perception we assume that we - as nucleated multi-cellular organisms - are more "advanced" than a single celled bacterium. In fact both humans and E.coli are the products of 4.5 billion years of evolution on planet Earth. Both are exquisitely adapted for the niches they occupy - both are at the pinnacle of evolutionary innovation.

Sentience is something new and probably emerged with primate mammals no more than 20 million years ago. As the genome shows, we have 98% commonality with mice, which manifestly do not demonstrate sentience. Sentience is not easily described in terms of metabolomics, proteomics or genomics. Yet - human sentience is written in the one-dimensional chain of nucleotides that is our genome.

Demotion for DNA
The title of this piece is DNA makes RNA makes protein. I will not attempt to destroy this hierarchy, but I have to say that I am no longer happy to accord DNA its CEO status. In fact I have to say that I rate DNA as a company would rate its archivist, vital in the long term, irrelevant for most day to day business and clearly subordinate to overall corporate strategy. Indeed too much respect for "the way things have been done" can be a constraint in changing times. Evolution challenges DNA with mutations which alter the company records!

The Genome
A few weeks ago the human genome followed viruses, bacteria and plants into the realm of "the known". That is to say a near complete physical description of the sequence of about 6 billion nucleotide residues in the DNA of 24 chromosomes (22 autosomes plus X and Y) contributing to the diploid component of 46 chromosomes (44XX female or 44XY male) has been published.

That sequence in itself is nothing. What is important is how that sequence determines the proteins (and RNAs) that permit biochemistry. The genome contains genes or inheritable elements that do something. The genes are the verbs of the language of life. Somewhat to our surprise the language of human life makes do with about 30,000 verbs. We thought that our magnificence required a greater vocabulary! The verb analogy is not too bad, for like verbs, genes can be used in different constructions leading to differing outcomes.

The challenge of genomics is now to understand how the 30,000 genes are influenced by the roughly 98% of the genome which does not code for proteins or functional RNA. Personally, I am no longer sure what the word "gene" means. I cannot believe that 98% of the genome, which is - more or less -  faithfully inherited, is without function. Function, to my naive mind means genes. It may be that we must revise our concept of what constitutes a gene. A gene may not necessarily be a contiguous piece of DNA coding for a derived  linear protein or RNA sequence. "Pick and mix" options may surprise us.

The transcriptome
DNA is copied as RNA in a process called transcription. RNA is almost identical to DNA, except that:

  1. a labile hydroxyl-carbon bond (C-OH) replaces the more stable hydrogen-carbon bond (C-H) on the ribose sugar, which forms the nucleic acid backbone.
  2. the nucleoside uracil replaces thymidine (which has an extra CH3 group in place of a hydrogen on the number 5 Carbon in carbon-nitrogen pyrimidine ring

The RNA transcript of the master DNA template is subject to editing. This editing can be radical (as in nucleated mammalian cells) or relatively trivial as in some bacterial cells. In the eukaryote, editing occurs in two or three stages, firstly major editing inside the nucleus and at the nuclear membrane, and subsequently in the cytoplasm. Bacterial mRNAs tend to be long, coding for several genes (polycistronic), whereas eukaryote mRNAs tend to code for just one protein sequence (monocistronic). In the eukaryote cell nucleus information carrying DNA (genes) is usually interspersed with other DNA sequences known as introns. These introns are edited out of the RNA transcript released into the cytoplasm. Nuclear RNA processing involves both protein and RNA-based enzymatic activities. Prokaryotic DNA generally contains fewer introns, although this is complicated as the different prokaryote kingdoms are quite different (the Archaeal bacteria have resemblances - including introns - to eukaryotes).

Several different classes of RNA are found in the cytoplasm of both eukaryote and prokaryote cells. We will not look closely at nuclear RNAs here (a good text on molecular biology or cell biology will offer guidance).

The eukaryotic ribosome is synthesised in the nucleus, in a structure known as the nucleolus, and is exported to the cytoplasm as partially assembled protein/rRNA complexes. Ribosome synthesis and nucleolar chemistry are a special subset of both the transcriptome and the proteome.

As the above lines demonstrate the transcriptome is not simple!
 

The Proteome
This word is used to describe the entirety of proteins expressed by a given cell. In a complex organism, different cells and tissues may express different proteins. The proteins present in a brain cell are not the same as those present in a liver cell - although a great many will be conducting identical "housekeeping" functions.  In any organism or cell, protein expression may vary depending upon circumstances or external signals. Immature proteins are synthesised on mature ribosomes, which in both prokaryotes and eukaryotes are found in the cytoplasm. Ribosomes may be soluble or membrane attached.

Immature proteins may be subject to a variety of post-translational modifications. These may include:

The study of the proteome (or proteomics), will yield important new understanding of disease states and potential therapeutic approaches. Documenting the total proteome, or expressed protein complement of a cell, tissue or organism is a non-trivial task. For almost 25 years, 2-dimensional acrylamide gels (iso-electric focussing versus SDS electrophoresis) have provided the best insight to protein complements. Enhanced protein resolution and sensitive radio-labelling and staining techniques have taken 2D gels almost to the limits of exploitation. The marriage of electrophoretic and chromatographic techniques with mass spectrometry is now adding new insight to the proteome and new companies based on variations of these approaches are now flourishing.

The Metabolome
A glance at any metabolic pathways chart will give a superficial understanding of the complexity of chemistry in the living cell. The organic molecules which constitute intermediary metabolism vary significantly from species to species. Plants have made a speciality of synthesising complex alkaloids which they use to defend themselves. Some animals have evolved potent toxins based on short chains of amino acids. Much of the complexity of metabolism is subordinated to the requirements of housekeeping. Energy must be provided from a range of substrates. Simple molecules must be assembled into the building blocks for proteins and nucleic acids. membranes must be synthesised to keep the inside in and the outside out. As well as de novo synthesis, several key molecules must be subject to repair and maintenance (for example DNA). The metabolome is dynamic. If it is in any way stressed it will adopt a configuration which absorbs that stress. Homeostatic mechanisms can be short and long term. Rapid responses to some stimuli might be complemented by more long-term activation of gene expression.

Although protein expression can give some insight into the chemistry of the cell, it cannot accurately indicate the complex of interacting chemical pathways. A potentially infinite level of complexity is reduced to manageable proportions by the process of compartmentalisation. In both time and physical space chemical processes are tidied up into finite compartments. Control and co-ordination of metabolism would be near to impossible in a single compartment cell. Macromolecular complexes, membrane systems, ionic gradients and diffusion processes all serve to structure the cytoplasm in cells. Time further serves to separate reactive components until the appropriate moment for coming together.
 

The Wholome?
No the word does not exist! However even the most esoteric chemistry of the strangest cell is implicit in the one dimensional DNA sequence of the genome. Other than nucleic acids and proteins, no chemical entities are directly under the control of the genome. Control is indirect and the result of multi-enzyme metabolic pathways and the specific availability of appropriate precursors.

The reduction to compartments and the increasing insight into the nature of homeostatic processes at cellular level is giving us hope that the number of variables in a living cell is very large but finite. Computers can today handle huge complexity and this gives hope that one day we might make better and better models of cellular function and chemistry.
 

DIAGRAM SHOWING INFORMATION FLOW IN A EUKARYOTIC CELL (Repeat).

At every stage, within the cell, feedback processes are in operation. Biochemistry is self regulating. Note that RNA, more than DNA appears to play a leading role in information flow. Proteins and the proteome have perhaps the most profound functional impact. The metabolome is more diffuse, yet also more linked with the environment, be it surrounding cells or the ecology of which the cell is a part. The outer cell membrane links both metabolome and proteome to the outside.

 

 

 

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