Tillmann Pape

Single-particle electron cryo-microscopy: towards atomic resolution

4. Single particles and angular reconstitution 323 4.1 Preliminary filtering and centring of data 323 4.2 Alignments using correlation functions 324 4.3 Choice of first reference images 324 4.4 Multi-reference alignment of data 325 4.5 MSA eigenvector/eigenvalue data compression 328 4.6 MSA classification 330 4.7 Euler angle determination (‘ angular reconstitution ’) 332 4.8 Sinograms and sinogram correlation functions 332 4.9 Exploiting symmetry 335 4.10 Three-dimensional reconstruction 337 4.11 Euler angles using anchor sets 339 4.12 Iterative refinements 339

Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.

The kinetic mechanism of elongation factor Tu (EF‐Tu)‐dependent binding of Phe‐tRNAPhe to the A site of poly(U)‐programed Escherichia coli ribosomes has been established by pre‐steady‐state kinetic experiments. Six steps were distinguished kinetically, and their elemental rate constants were determined either by global fitting, or directly by dissociation experiments. Initial binding to the ribosome of the ternary complex EF‐Tu·GTP·Phe‐tRNAPhe is rapid (k1 = 110 and 60/μM/s at 10 and 5 mM Mg2+, 20°C) and readily reversible (k−1 = 25 and 30/s). Subsequent codon recognition (k2 = 100 and 80/s) stabilizes the complex in an Mg2+‐dependent manner (k−2 = 0.2 and 2/s). It induces the GTPase conformation of EF‐Tu (k3 = 500 and 55/s), instantaneously followed by GTP hydrolysis. Subsequent steps are independent of Mg2+. The EF‐Tu conformation switches from the GTP‐ to the GDP‐bound form (k4 = 60/s), and Phe‐tRNAPhe is released from EF‐Tu·GDP. The accommodation of Phe‐tRNAPhe in the A site (k5 = 8/s) takes place independently of EF‐Tu and is followed instantaneously by peptide bond formation. The slowest step is dissociation of EF‐Tu·GDP from the ribosome (k6 = 4/s). A characteristic feature of the mechanism is the existence of two conformational rearrangements which limit the rates of the subsequent chemical steps of A‐site binding.

Induced fit in initial selection and proofreading of aminoacyl‐tRNA on the ribosome

The fidelity of aminoacyl‐tRNA (aa‐tRNA) selection by the bacterial ribosome is determined by initial selection before and proofreading after GTP hydrolysis by elongation factor Tu. Here we report the rate constants of A‐site binding of a near‐cognate aa‐tRNA. The comparison with the data for cognate aa‐tRNA reveals an additional, important contribution to aa‐tRNA discrimination of conformational coupling by induced fit. It is found that two rearrangement steps that limit the chemical reactions of A‐site binding, i.e. GTPase activation (preceding GTP hydrolysis) and A‐site accommodation (preceding peptide bond formation), are substantially faster for cognate than for near‐cognate aa‐tRNA. This suggests an induced‐fit mechanism of aa‐tRNA discrimination on the ribosome that operates in both initial selection and proofreading. It is proposed that the cognate codon‐anticodon interaction, more efficiently than the near‐cognate one, induces a particular conformation of the decoding center of 16S rRNA, which in turn promotes GTPase activation and A‐site accommodation of aa‐tRNA, thereby accelerating the chemical steps. As kinetically favored incorporation of the correct substrate has also been suggested for DNA and RNA polymerases, the present findings indicate that induced fit may contribute to the fidelity of template‐programed systems in general.

Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome.

Binding of aminoglycoside antibiotics to 16S ribosomal RNA induces a particular structure of the decoding center and increases the misincorporation of near-cognate amino acids. By kinetic analysis we show that this is due to stabilization of the near-cognate codon recognition complex and the acceleration of two rearrangements that limit the rate of amino acid incorporation. The same rearrangement steps are accelerated in the cognate coding situation. We suggest that cognate codon recognition, or near-cognate codon recognition augmented by aminoglycoside binding, promote the transition of 16S rRNA from a ‘binding’ to a ‘productive’ conformation that determines the fidelity of decoding.

Structure of the Escherichia coli ribosomal termination complex with release factor 2

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein–transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the ‘SPF’ loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.

Initial Binding of the Elongation Factor Tu·GTP·Aminoacyl-tRNA Complex Preceding Codon Recognition on the Ribosome

The first step in the sequence of interactions between the ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA, is the codon-independent formation of an initial complex. We have characterized the initial binding and the resulting complex by time-resolved (stopped-flow) and steady-state fluorescence measurements using several fluorescent tRNA derivatives. The complex is labile, with rate constants of 6 × 107M−1 s−1 and 24 s−1 (20°C, 10 mM Mg2+) for binding and dissociation, respectively. Both thermodynamic and activation parameters of initial binding were determined, and five Mg2+ ions were estimated to participate in the interaction. While a cognate ternary complex proceeds from initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognate complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases. Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA. This suggests that codon-anticodon recognition, which takes place after the formation of the initial binding complex, provides a specific signal that triggers fast GTP hydrolysis by EF-Tu on the ribosome.

Hexameric ring structure of the full‐length archaeal MCM protein complex

In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three‐dimensional reconstruction of the full‐length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring‐shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double‐stranded DNA. The crystal structure of the amino‐terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy‐terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double‐stranded DNA translocases.

Modus operandi of the bacterial RNA polymerase containing the σ54 promoter‐specificity factor

Bacterial sigma (σ) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative σ factor σ54 confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the σ54 factor makes with the bacterial transcription machinery.

Organization of an activator-bound RNA polymerase holoenzyme.

Summary Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor σ54 remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with σ54 alone, and of RNAP-σ54 with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-σ54 closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.

Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity

The MCM complex from the archaeon Methanother-mobacter thermautotrophicus is a model for the eukaryotic MCM2-7 helicase. We present electron-microscopy single-particle reconstructions of a DNA treated M.thermautotrophicus MCM sample and a ADP·AlFx treated sample, respectively assembling as double hexamers and double heptamers. The electron-density maps display an unexpected asymmetry between the two rings, suggesting that large conformational changes can occur within the complex. The structure of the MCM N-terminal domain, as well as the AAA+ and the C-terminal HTH dom-ains of ZraR can be fitted into the reconstructions. Distinct configurations can be modelled for the AAA+ and the HTH domains, suggesting the nature of the conformational change within the complex. The pre-sensor 1 and the helix 2 insertions, important for the activity, can be located pointing towards the centre of the channel in the presence of DNA. We propose a mechanistic model for the helicase activity, based on a ligand-controlled rotation of the AAA+ subunits.