Friday, 24 February 2012

Beyond the Central Dogma

Biological information may be represented in different ways. The famous central dogma of molecular biology, as described by Francis Crick and others in the 1970s, states that "DNA makes RNA makes protein" (see this page in PPS section 6: Bioinformatics).. Nowadays, some exceptions to this simple statement of the single-direction flow of information are known, such as the synthesis of DNA molecules from a RNA template by the retroviral reverse transcriptases or the synthesis of telomeres by telomerase. However, it is very likely that early biology used a simpler molecular architecture. The "RNA World" hypothesis states that early life was based on RNA alone - the only molecule that is capable of both storing information and catalysing chemical reactions - and was only later superseded by  the DNA-RNA-protein world of today.
Such an RNA world would have relied on an ability of RNA molecules, or "ribozymes" to synthesise other RNAs following a template: another way of saying that in this world, each of the components of the "central dogma" would have been represented by a type of RNA. Phillip Holliger, a group leader in the Protein and Nucleic Acid Chemistry division of the MRC Laboratory of Molecular Biology, Cambridge, gave an interesting seminar to the Institute of Structural and Molecular Biology at UCL in early February in which he described his recent work exploring how the chemistry of this "RNA World" might have worked.

Holliger began by introducing the concept of a RNA replicase: an RNA sequence that can catalyse the extension of a RNA primer. No natural RNA replicases are known: if there was once such a primordial molecule, it has been lost in time. In order to understand the RNA replicase, therefore, it is first necessary to synthesise one.  In 2001, David Bartel and his group at MIT in Boston, Massachusetts, published a paper (Johnston et al. (2001), Science 292, 1319-25) describing a synthetic ribozyme that was able to catalyse the addition of over a complete turn of a RNA primer strand on a template sequence to a "reasonable" level of accuracy.  Holliger's group is one of several that have, since then, been making improvements to the basic replicase.

The work in the Holliger group has involved developing a technique known as compartmentalized bead-tagging (CBT)  for directing the "evolution" of a synthetic RNA polymerase. Put very simply, this involves using water-in-oil emulsions to select and isolate ribozymes from a library that had specific RNA primer extension properties. Observing that their original ribozymes had poor ribozyme-template-primer interactions, they generated a library of ribozymes with additional random 5' domains. Three rounds of CBT were needed to isolate a ribozyme named C19 that had improved RNA polymerase activity. Secondary structure prediction suggested that the new 5' domain of this ribozyme consisted of a short sequence complementary to the 5' end of the RNA template used in the experiments, followed by a hairpin domain. This sequence complementarity promotes the formation of a stable ternary complex between the ribozyme and the template and primer RNA strands thereby allowing processive synthesis of long RNA molecules. Further directed mutations have yielded a ribozyme (tC19Z) that can catalyse the synthesis of a RNA that is itself catalytic: a mini-version of a hammerhead ribozyme (link to PDB structure 1MME).. Holliger's group may still be quite a long way from generating a truly self-replicating molecule (which would have been necessary in a primordial RNA world) but they are making progress towards this goal.

This work can essentially be seen as "restricting" or "shrinking" the central dogma to one type of  information-containing macromolecule from three. Holliger and his group are now using similar techniques to try to "expand" the dogma by adding an extra branch, developing polymerases that can synthesise and reverse transcribe artificial poly-nucleotides based on unnatural building blocks not found in nature.  

Holliger's work on the ribozymes was published last year in the journal Science: Wochner et al. (2011), Science 332, 209-212. Click here to access this paper (login to Birkbeck e-library required).