IGEM:IMPERIAL/2007/Tutorials/Guide for Engineers/Central Dogma of Molecular Biology

Introduction

“The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.” - Francis Crick, 1958

The Central Dogma

It might seem like a whole load of jargon, but the basic concept underlying the central dogma is that the flow of information proceeds in a linear step-wise fashion, from DNA to RNA and finally protein. This means that protein information cannot flow back to that of nucleic acids. The central dogma is a fundamental concept that basically forms the structural framework with which molecular biologists understand the transfer of sequence information between the different biopolymers.

So why is the central dogma important to synthetic biology? Many recombinant DNA technologies, as with our understanding of cellular processes, are more often than not based or adapted from what we know. Knowledge is power, and the more we understand, the more we are able to manipulate its machinery to meet our required specifications. And we do know a lot about the mechanisms behind it.

The Basics: Cell Organization and DNA Structure

The cell is a compartment (not necessarily the simplest form of a living system), and has the ability to localize concentration of metabolites, substrates, and catalysts. Cells are always bounded by an amphipathic lipid bilayer, usually made of phospholipids.

General similarities:

• Proteins: L-amino acids (common chirality – D-amino acids are very rare – peptidoglycan).
• Carbohydrates: glucose/glycolysis as main energy source, α-(1→4) glucans for energy storage, β-(1→4) glucans (cellulose, chitin) for structural purposes.
• RNA: universality of the genetic code, all cells possess very similar ribosomes.
• DNA: almost invariably the genetic material.
• Lipids: terpenes and phospholipids used for membranes.
• Porphyrins: haem, chlorophyll, bilin pigments, all with tetrapyrrole motif. Used for redox (electrons, oxygen, etc.).

Prokaryotes

Representative Graphic of a Prokaryotic cell

Prokaryotes have a simple cell morphology, and are characterized by the lack of a nucleus, leaving a free circular genophore.

Other properties:

• ‘Simple’ rotating motor-type flagellum.
• Cell division by fission, genophore attached to plasmalemma by mesosome.
• Few membrane-bound organelles, no double-membrane bound organelles.
• They are ‘small’ because diffusion limits the rate of transport across the cell – 1 μm.

Prokaryotic DNA also lacks histones (packaging proteins) and molecules have direct access to DNA. They have a junk-free genome, transcribed by just one RNA polymerase. Transcription leads to translation with no intermediate processing, which allows several genes to be encoded in a single transcriptional unit (a polycistron). They have small (70S) ribosomes.

Eukaryotes

Representative Graphic of a Eukaryotic cell

Eukaryotes have a complex system of internal organelles (although not necessarily more complex by nature), and is characterized by the presence of a nucleus with its genophore enclosed within.

Other properties:

• Double-membrane bound nucleus containing linear chromosomes.
• Complex 9+2-type undulipodium, cytoskeleton, cytosis and mitosis.
• Many membrane-bound organelles and double membrane-bound endosymbionts (e.g. mitochondria, chloroplasts).
• They can grow ‘large’ because cytoplasmic streaming allows rapid transport across the cell – 100 μm.

Eukaryotic DNA is bound by histones, and requires some degree of unpacking for expression. Much of their genome is composed of parasitic DNA and introns. Three RNA polymerases exists, (approximately) one for each sort of major RNA product:

• RNApol-I – rRNA.
• RNApol-II – mRNA and snRNA.
• RNApol-III – tRNA and 5S rRNA.

RNA is heavily processed in the nucleus (e.g. splicing mechanisms). They possess larger 80S ribosomes.

Structure of DNA

Structure of DNA

Protein Trafficking and Cell-cell Communications

Lipid Bilayer

Most biological molecules are impermeable to the lipid bilayer based on size and charge. Exception applies to very small uncharged molecules (eg. water, carbon dioxide, nitrogen), or small signaling molecules such as acyl homoserine lactone (AHL) in quorum sensing and steroid hormones in humans.

Protein Trafficking

Channels are required to transport molecules across the membrane, and are usually specific to a certain molecule - sometimes even specific to the direction of transport (only import or export). Some channels transport two molecules together, using the potential of one molecule to assist the transport of the second molecule.

Channels can also be classified as passive (allows diffusion only) or active (uses energy to transport against the concentration gradient), and must also be able to open and close to control the flux.

Proteins are usually very large molecules and thus cannot diffuse through the lipid bilayer or use channels. Proteins translated in the cells are usually not folded prior to export, and signal sequences are added as extra bits of translated protein that tags it for export. The membrane transporters then recognize these tags and transport tagged proteins out of the cell.

Quorum Sensing

Quorum sensing is a type of autocrine signaling, and is used by Vibrio fischeri to release light at high cell counts.

The lux operon is regulated by 2 proteins:

• luxR (lux regulator gene) is transcribed constitutively to produce LuxR. LuxR can only stimulate translation of luxpR (lux promoter) in the presence of molecule acyl homoserine lactone (AHL).
• luxI (lux inducer gene) is transcribed at basal (low) levels, producing low levels of LuxI. LuxI, is an enzyme that converts S-adenosylmethionine (SAM) into AHL.

Low Cell Density

At low AHL concentration, there are low levels of LuxR-AHL complex formation, and thus low levels of transcription of luxpR.

High Cell Density

At high AHL concentration, more cells are able to produce LuxI and AHL, thereby leading to more LuxR-AHL complex formed. This allows increased activation of luxpR, leading to a higher transcription rate of luxI. As more LuxI is generated, it acts in a positive feedback to the system to generate more AHL.

Downstream genes (luxCDABE) code of luciferase (an actuator) which generates light.

Therefore, at high cell density, light is generated.