Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE.

Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome , initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE® technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome . This study provides a proof of concept for the application of this method on large complexes.

ITC Studies of Ribosome/Antibiotics Interactions.

The fight against multiresistant bacteria responsible for nosocomial diseases has recently been classified as an absolute priority by the World Health Organization. For some organisms, priority status has even been assessed as critical, as almost all currently available antibiotics are now inefficient against these "super-bacteria." Ribosome is a major target of several antibiotics, and extensive biochemical and structural studies led to a better understanding of the mechanism of action of drugs targeting translation (Blair et al., Nat Rev Microbiol 13:42-51, 2015; Lin et al., Annu Rev Biochem 87:451-478, 2018; Wilson, Nat Rev Microbiol 12:35-48, 2014; Yonath, Annu Rev Biochem 74:649-79, 2005). However, our knowledge regarding thermodynamic data of compounds targeting the ribosome, which are yet essential for a complete understanding of translation inhibition mechanisms by drugs, is still very poor.In this chapter we describe the use of ITC microcalorimetry to investigate the binding of bacterial ribosome to two antibiotics targeting the peptide tunnel: macrolides and proline-rich antimicrobial peptides (PrAMPs). This strategy yields reliable and artifact-free binding parameters for antibiotics and provides an original view on ribosome/antibiotics interactions.

Thermodynamics of Molecular Machines Using Incremental ITC.

Molecular biomachines, such as DNA and RNA polymerases or the ribosome, are fascinating biological assemblies able to swiftly perform repeated and highly regulated tasks, with a remarkable accuracy. Significant advances in structural studies during the past 20 years provided a wealth of information regarding their architecture and considerably contributed to a better understanding of their mechanism of action. However, the three-dimensional structure of a biological nanomachine alone does not provide access to its detailed mechanism of action, even when obtained at atomic resolution. When combined with other biophysical approaches, thermodynamic data, together with kinetic data, are essential for a complete description of any binding interaction, revealing forces driving complex formation and providing insights into mechanisms of action. We have developed an incremental ITC approach that is well-suitable for analysis of biomolecular machines. This strategy allows a dissection of molecular biomachine reactions through successive additions in the ITC cell of consecutive substrates.

Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors.

HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.