Structural analysis of stalled ribosomal complexes and their respective rescue mechanisms by Cryo-Electron Microscopy

The ribosome is a multifunctional ribonucleoprotein complex responsible for the translation of the genetic code into proteins. It consists of two subunits, the small ribosomal subunit and the large ribosomal subunit. During initiation of translation, both subunits join and form a functional 70S ribosome that is capable of protein synthesis. In the course of elongation, the ribosome synthesizes proteins according to the codons on the mRNA until it encounters a stop codon leading to the recruitment of release factors 1 or 2 followed by release of the nascent chain. Upon release of the polypeptide chain the subunits dissociate from each other and can be recruited for another round of translation. There are two scenarios that interfere with active translation, namely the formation of so called ‘non-stop’ or ‘no-go’ complexes. In both cases, ribosomes pause translation and without interference of additional factors, they would become stalled. Accumulation of such events leads to a decrease of ribosomal subunits that can be recruited for translation, ultimately resulting in the death of the cell. Using cryo-electron microscopy (cryo-EM), we obtained the structure of alternative rescue factor A (ArfA) together with release factor 2 bound to a ‘non-stop’ complex. Our reconstructions showed that the C-terminal domain of ArfA occupies the empty mRNA channel on the SSU, whereas the N-terminal domain provides a platform for recruiting RF2 in a stop codon-independent way. Thereby, ArfA stabilizes a unique conformation of the switch loop of RF2, responsible for directing the catalytically important GGQ motif towards the PTC. The high-resolution structure of ArfA allowed us to compare its mode of action with trans-translation and alternative rescue factor B, two other factors operating on ‘non-stop’ complexes. A second project focused on elongation factor P (EF-P), a factor that alleviates stalling on polyproline stalled ribosomes. Applying cryo-EM, we were able to show that in the absence of EF-P, the nascent chain is destabilized as the polyproline moiety attached to the P-tRNA is not able to accommodate within the ribosomal tunnel. Binding of modified EF-P to the polyproline stalled complex stabilizes the P-site tRNA and especially the CCA, thereby forcing the nascent chain to adopt an alternative conformation that is favorable for translation to proceed

Structural analysis of stalled ribosomal complexes and their respective rescue mechanisms by Cryo-Electron Microscopy

The ribosome is a multifunctional ribonucleoprotein complex responsible for the translation of the genetic code into proteins. It consists of two subunits, the small ribosomal subunit and the large ribosomal subunit. During initiation of translation, both subunits join and form a functional 70S ribosome that is capable of protein synthesis. In the course of elongation, the ribosome synthesizes proteins according to the codons on the mRNA until it encounters a stop codon leading to the recruitment of release factors 1 or 2 followed by release of the nascent chain. Upon release of the polypeptide chain the subunits dissociate from each other and can be recruited for another round of translation. There are two scenarios that interfere with active translation, namely the formation of so called ‘non-stop’ or ‘no-go’ complexes. In both cases, ribosomes pause translation and without interference of additional factors, they would become stalled. Accumulation of such events leads to a decrease of ribosomal subunits that can be recruited for translation, ultimately resulting in the death of the cell. Using cryo-electron microscopy (cryo-EM), we obtained the structure of alternative rescue factor A (ArfA) together with release factor 2 bound to a ‘non-stop’ complex. Our reconstructions showed that the C-terminal domain of ArfA occupies the empty mRNA channel on the SSU, whereas the N-terminal domain provides a platform for recruiting RF2 in a stop codon-independent way. Thereby, ArfA stabilizes a unique conformation of the switch loop of RF2, responsible for directing the catalytically important GGQ motif towards the PTC. The high-resolution structure of ArfA allowed us to compare its mode of action with trans-translation and alternative rescue factor B, two other factors operating on ‘non-stop’ complexes. A second project focused on elongation factor P (EF-P), a factor that alleviates stalling on polyproline stalled ribosomes. Applying cryo-EM, we were able to show that in the absence of EF-P, the nascent chain is destabilized as the polyproline moiety attached to the P-tRNA is not able to accommodate within the ribosomal tunnel. Binding of modified EF-P to the polyproline stalled complex stabilizes the P-site tRNA and especially the CCA, thereby forcing the nascent chain to adopt an alternative conformation that is favorable for translation to proceed

Atomic mutagenesis at the ribosomal decoding site

<p>Ribosomal decoding is an essential process in every living cell. During protein synthesis the 30S ribosomal subunit needs to accomplish binding and accurate decoding of mRNAs. From mutational studies and high-resolution crystal structures nucleotides G530, A1492 and A1493 of the 16S rRNA came into focus as important elements for the decoding process. Recent crystallographic data challenged the so far accepted model for the decoding mechanism. To biochemically investigate decoding in greater detail we applied an <i>in vitro</i> reconstitution approach to modulate single chemical groups at A1492 and A1493. The modified ribosomes were subsequently tested for their ability to efficiently decode the mRNA. Unexpectedly, the ribosome was rather tolerant toward modifications of single groups either at the base or at the sugar moiety in terms of translation activity. Concerning translation fidelity, the elimination of single chemical groups involved in a hydrogen bonding network between the tRNA, mRNA and rRNA did not change the accuracy of the ribosome. These results indicate that the contribution of those chemical groups and the formed hydrogen bonds are not crucial for ribosomal decoding.</p

The role of the prefrontal cortex in maintenance and interference control processes of working memory assessed with multi-channel near-infrared spectroscopy

Egetemeir J, Huter TJ, Pauli P, Fallgatter AJ, Herrmann MJ. The role of the prefrontal cortex in maintenance and interference control processes of working memory assessed with multi-channel near-infrared spectroscopy. Journal of Psychophysiology. 2006;20(2):139-140

Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ(2) mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery)