Escherichia coli CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression.

Escherichia coli CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of cspA mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the cspA mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

Efficient production of protein complexes in mammalian cells using a poxvirus vector.

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFß, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

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.