Structural Studies of RNA-dependent RNA polymerases

Most RNA viruses possess an RNA-dependent RNA polymerase (vRdRP), responsible for viral genome replication and transcription. Furthermore, a number of eukaryotic organisms, including plants, fungi, protozoa and some metazoans, produce cellular RdRPs (cRdRPs) involved in RNA silencing mechanisms. One of the best studied vRdRPs is that of the dsRNA bacteriophage Φ6. Structures of Φ6 RdRP (Φ6pol) in complex with RNA oligonucleotides revealed the basis for template specificity: the extra hydroxyl group leads to additional RNA-protein interactions, further stabilizing the template. Structures of "manganese-free" Φ6pol and a mutated form of the protein with lower affinity for the ion (E491Q mutant) provided some hints to the role of manganese. The structure of a complex of Φ6pol with RNA oligonucleotides and GTP with the catalytic magnesium ions substituted by calcium ions shows a distorted geometry of the initiation competent state, providing a molecular explanation of the calcium inhibitory effect. Finally, the structure of a mutated form of the protein (628QYKW632-SG mutant) prone to back-priming initiation revealed a set of contacts important for de novo initiation. Considering the high structural homology of Φ6pol with other vRdRPs, particularly from (+)ssRNA Hepatitis C Virus (HCV), insights into the mechanistic and structural details of Φ6pol are thought to be relevant to the general understanding of vRdRPs. The dimeric structure of QDE-I, an RdRP from the fungus Neurospora crassa involved in RNA silencing, revealed a surprising similarity at the active site level to multi subunit DNA-dependent RNA polymerases (DdRPs). This implies a close evolutionary relationship between these erzymes and a possible connection between RNA silencing pathways and primordial RNA polymerisation mechanisms. Furthermore, an analysis based on the structures of several template dependent polymerases suggests that they have emerged more than once over the course of evolution

A semiotic analysis of the genetic information

Terms loaded with informational connotations are often employed to refer to genes and their dynamics. Indeed, genes are usually perceived by biologists as basically ‘the carriers of hereditary information.’ Nevertheless, a number of researchers consider such talk as inadequate and ‘just metaphorical,’ thus expressing a skepticism about the use of the term ‘information’ and its derivatives in biology as a natural science. First, because the meaning of that term in biology is not as precise as it is, for instance, in the mathematical theory of communication. Second, because it seems to refer to a purported semantic property of genes without theoretically clarifying if any genuinely intrinsic semantics is involved. Biosemiotics, a field that attempts to analyze biological systems as semiotic systems, makes it possible to advance in the understanding of the concept of information in biology. From the perspective of Peircean biosemiotics, we develop here an account of genes as signs, including a detailed analysis of two fundamental processes in the genetic information system (transcription and protein synthesis) that have not been made so far in this field of research. Furthermore, we propose here an account of information based on Peircean semiotics and apply it to our analysis of transcription and protein synthesis

Life’s order, complexity, organization, and its thermodynamic–holistic imperatives

In memoriam Jeffrey S. Wicken (1942–2002)—the evolutionarily minded biochemist, who in the 1970/80s strived for a synthesis of biological and physical theories to fathom the tentative origins of life. Several integrative concepts are worth remembering from Wicken’s legacy. (i) Connecting life’s origins and complex organization to a preexisting physical world demands a thermodynamically sound transition. (ii) Energetic ‘charging’ of the prebiosphere must precede the emergence of biological organization. (iii) Environmental energy gradients are exploited progressively, approaching maximum interactive structure and minimum dissipation. (iv) Dynamic self-assembly of prebiotic organic matter is driven by hydrophobic tension between water and amphiphilic building blocks, such as aggregating peptides from non-polar amino acids and base stacking in nucleic acids. (v) The dynamics of autocatalytic self-organization are facilitated by a multiplicity of weak interactions, such as hydrogen bonding, within and between macromolecular assemblies. (vi) The coevolution of (initially uncoded) proteins and nucleic acids in energy-coupled and metabolically active so-called ‘microspheres’ is more realistic as a kinetic transition model of primal biogenesis than ‘hypercycle replication’ theories for nucleic acid replicators on their own. All these considerations blend well with the current understanding that sunlight UV-induced photo-electronic excitation of colloidal metal sulfide particles appears most suitable as a prebiotic driver of organic synthesis reactions, in tight cooperation with organic, phase-separated, catalytic ‘microspheres’. On the ‘continuist vs. miraculist’ schism described by Iris Fry for origins-of-life considerations (Table 1), Wicken was a fervent early protagonist of holistic ‘continuist’ views and agenda

Mechanisms contributing to RNA localization and translation: study of neuronal zipcodes in primary cortical neurons, and translational changes in neurodegeneration

The regulation of gene expression in terms of space and time is governed by the distribution of RNA and proteins within a cell. This tightly controlled regulation is necessary for mediating cellular development and function. The localization of RNA is also a highly controlled process that is influenced by specific cis and trans-acting elements. In polarized cells, such as neurons, the localization of transcripts towards axons and dendrites (neurites) enables the immediate and efficient local synthesis of proteins in response to external stimuli. This thesis focuses on the examination of the cis-elements, or "zipcodes," that contribute to the localization of RNA towards the neurites and the translational defects that result in the peripheral neuropathy Charcot-Marie-Tooth (CMT) disease