Strukturaufklärung Ribosomaler Komplexe mittels Kryo-EM Mechanismus der Translokationsreaktion ribosomal verankerte ncRNAs

Die am Ribosom erfolgende Proteinbiosynthese unterteilt sich in die vier Schritte Initiation, Elongation, Termination und Recycling. Die Elongationsphase als zyklisch ablaufender Vorgang beinhaltet die Anlieferung aminoacylierter tRNAs in die A-Stelle des Ribosoms, die Bildung einer neuen Peptid...

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Bibliographic Details
Main Author: Ratje, Andreas Heinrich
Contributors: Hartmann, Roland K. (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2011
Pharmazeutische Chemie
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Table of Contents: Protein biosynthesis is carried out by the ribosome and can be divided into four steps: initiation, elongation, termination and recycling. The elongation phase, a cyclic process, comprises the delivery of aminoacylated tRNAs to the A-site, peptide bond formation and translocation of tRNAs together with bound mRNA. In prokaryotes, translocation is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. The exact mechanism of translocation is still unclear, although many biochemical studies have addressed this functional aspect of translation and high resolution structures of ribosomal complexes containing EF-G have become available. The intricate interplay of EF-G, tRNAs and different conformational states of the ribosome during translocation is far from being fully understood in mechanistic terms. As a result of this PhD work, novel insight into the mechanism of translation has been gained. Starting point was the formation and purification of ribosomal complexes consisting of 70S ribosomes from T. thermophilus bound to EF-G in its GDP form stalled with fusidic acid; the antibiotic allows GTP hydrolysis but inhibits conformational changes of EF-G and dissociation of EF-G from the ribosome. Such particles, further containing a deacylated tRNA, were analyzed by cryo-electron microscopy (cryo-EM). The collected cryo-EM data could be divided by multiparticle refinement into two, as yet undetected subpopulations within EF-G:ribosome complexes with 7.8 Å resolution for the first and 7.6 Å resolution for the second subpopulation. Both subpopulations show density for EF-G and one tRNA, but represent different conformational states. The first subpopulation showed a 50S:30S ratcheting of 7 with a modest swiveling of the 30S head of 5. In contrast, a smaller ratcheting of only 4 is observed for the second subpopulation, but in this case the swivelling of the 30S head is increased to 18. The high resolution of the structures enabled the visualization of secondary structures and the generation of molecular models with the new developed algorithm MDFIT which allows for the flexible docking of crystal structures considering molecular flexibilities. It could be shown that the conformation of the first subpopulation is comparable to ribosomes in the PRE-state, with the tRNA in a hybrid position between P- and E-site (P/E) as described in the literature. Accordingly, this subpopulation has been proposed to represent a pre-translocational intermediate (TIPRE). The second subpopulation shows the ribosome and the tRNA in a new conformation. In comparison with the TIPRE state, the anticodon stem-loop (ASL) has moved about 8 10 °A towards the E-site while maintaining contact with P-site components of the 30S head, enabled by the tRNA following the movement of the head. The simultaneous interaction with P-site components of the 30S head and E-site components of the 30S body/platform is the hallmark of this newly identified intersubunit hybrid site within the small subunit. As the contact with the E-site of the 50S subunit is maintained, the previous nomenclature describing hybrid sites has to be extended. This new tRNA binding state is referred to as the pe/E hybrid position: the tRNA forms P-site contacts with the 30S head and E-site contacts with the 30S platform, while being positioned at the E-site on the 50S side. The ASL together with the bound mRNA is very close to a fully translocated tRNA and the conformation of the partially translocated tRNA is considered to be a post-translocational intermediate (TIPOST). In contrast to the twisted conformations of the tRNA, EF-G shows only slight positional differences within both subpopulations. A significant difference is seen for domain IV of EF-G which gets in contact with helix 34 of 16S rRNA in the TIPOST state. This contact is made possible by a small shift of the position of EF-G and by helix 34 following the movement of the head. The position of helix 34 has changed by 12 °A while interaction with the 530 region of 16S rRNA is disrupted. As a result, the mRNA entry channel is opened and translocation of the mRNA-tRNA2-complex is enabled. So far, intermediate states of inter-subunit rotation were considered to be intermediates on the pathway to the fully rotated state. The present findings show that movement of tRNAs and translocation is dependent on back-ratcheting of the subunits and swiveling of the 30S head. We propose that fast GTP hydrolysis influences the interaction of the switch I region of EF-G with the 30S subunit, leading to a destabilization of the fully ratcheted state to enable back rotation. Based on the model of the ribosome acting as a Brownian ratchet machine during translocation, EFG is suggested to act as a dynamic pawl, decoupling the unratcheting motions of the ribosome from the transition of hybrid state tRNAs back into their classical states. Thereby, EF-G provides directionality and accelerates translocation of the tRNAs via several intermediate inter- and intra-subunit hybrid states into the classical P/P and E/E sites of the POST state. The numerous non-coding RNAs (ncRNAs) discovered in recent years exert a plethora of biological functions. The knowledge of their three-dimensional structure is important towards understanding their mode of function. However, many ncRNAs have turned out to be not amenable to X-ray crystallography. Here we pursued the approach to express ncRNAs as part of the 23S rRNA in order to anchor and expose these ncRNAs on the structurally well-characterized ribosomal surface for structural analyses by cryo-electron microscopy. The use of cryo-electron microscopy combined with shockfreezing of the samples preserves the native state of the ncRNA and avoids generation of artefacts as they are possible in X-ray crystallography. Summary 101 Several different ncRNAs (RNase P RNA of E. coli, B. subtilis and T. thermophilus, 6S RNA of A. aeolicus and the ribozyme GIR1 of D. iridis) were covalently linked to distinct helices of 23S rRNA (helices 9, 25, 45 and 98) of E. coli to elucidate the structure of the inserted RNA molecule using the 70S ribosome as a platform of known structure. Expression of mutated ribosomes was explored in different E. coli strains using a heat-inducible promoter system. This approach required to separate the simultaneously expressed native ribosomes from the mutant ones. This was possible by purification via an MS2 tag which was inserted into helix 98 of mutated ribosomes. Using RT-PCR, it was impossible in most cases to detect significant levels of 23S rRNA carrying the ncRNA insertion, while detection of the MS2 tag was always possible in cr70S fractions representing a mixture of native and mutated ribosomes. Ribosomes purified via the MS2 tag were pure fractions of ribosomes carrying the MS2 tag, but the ncRNA insertions were hardly or not at all detectable. A distinct detection of mutant ribosomes carrying both the MS2 tag and the ncRNA was only feasible in the case of the ribozyme GIR1 inserted into helix 9 of 23S rRNA. The corresponding strain had to be induced for only 30 minutes, resulting in expression of mutated 23S rRNA to a level of 10 20% of total cellular 23S rRNA. Affinity purification of corresponding cr70S preparations resulted in ribosomes all carrying the MS2 tag in helix 98, but only 30% also harboured the ribozyme GIR1 in helix 9. In general, the additional insertion in helix 9 seems to disturb the function of the ribosome, leading to excision of the inserted ncRNA and/or degradation of mutant 23S rRNA as part of as yet unknown cellular defense mechanisms. Cryo-EM analysis of affinity-purified ribosomes resulted in a volume with additional density for helix 9 (ribozyme GIR1) and helix 98 (MS2 tag) in 45% of the particle images. The resolution of 18.4 °A achieved so far is too low for a detailed structural interpretation, but provides the basis for the collection of a data set that is sufficient to to generate a high resolution structure.