Gary Mark Skinner, PhD

"Single-Molecule studies of the Central Dogma"

Introduction

    The central dogma of molecular biology, as proposed by Francis Crick (Crick, 1958), provides a simple overview of the essential processes of gene expression.  That is that genetic information flows from DNA to an RNA intermediate thence to the final functioning protein (Fig. 1).  There have since been important additions to this scheme, i) The enzyme reverse transcriptase is able to transmit information from RNA to DNA e.g. in HIV and, ii) A class of protein known as prions, first described by Stanley Prusiner in 1982 (Prusiner, 1982), are able to transmit "information" between proteins.  These are the infectious agents responsible for certain brain-wasting diseases such as bovine spongiform encephalopathy (BSE) and the human form, Creutzfeldt-Jacobs disease.

Fig. 1. The central dogma - The hereditary information contained within DNA is converted, by RNA polymerase, into a corresponding sequence of RNA that is then converted into a sequence of amino-acids by the ribosome.  This polypeptide then folds into it's final three-dimensional struture to form the active protein.  The structure pictured here is that of bacteriophage T7 RNA polymerase.

    While the central dogma can be simply stated, the process itself is far from simple.  It consists of multiple interactions between proteins and nucleic acids with many levels of regulation.  Primarily, it can be broken down into two "core" steps, transcription and translation.  Transcription is accomplished by the enzyme RNA polymerase (RNAP) reading the sequence of bases of one strand of DNA and producing an intermediate copy of this information known as "messenger" RNA (mRNA).  Translation is the next step, accomplished the macromolecular complex known as the "ribosome".  In this step, the mRNA sequence is read by the ribosome 3 bases at a time, or codon, at a time and is converted into a sequence of amino-acids forming the polypeptide that ultimately folds to form the functioning protein element.

    The traditional methods for studying the elements of the central dogma involve bulk experiments involving ensembles of many molecules.  The problem with such systems, while they have been very powerful, is that important information is lost due to averaging of all states within the system.  The only way to solve this is to synchronize all molecules within the population.  Practically, this is very difficult and synchronization would be rapidly lost due to the stochastic nature of these steps; the solution is to observe the events of the central dogma at the single-molecule level.

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