Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process

Valle, Mikel; Sengupta, Jayati; Swami, N.K.; Grassucci, Robert A.; Burkhardt, Nils; Nierhaus, Knud H.; Agrawal, Rajendra K.; Frank, Joachim

doi:10.1093/emboj/cdf326

Cited by 286 publications

(183 citation statements)

References 63 publications

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“…The van Heel group suggested that this is mainly a distortion in the anticodon loop (144), which would be consistent with the conformational variability of the loop already known by a comparison of various crystal structures containing tRNA. However, the Frank group suggests that the distortion is higher in the body of tRNA (143), and a more recent structure at even higher resolution suggests that it is between the ASL and the D stem-loop (Figure 4a) (146).…”

Section: Cryo-electron Microscopy Structures Of the Ribosomementioning

confidence: 99%

Structural Insights Into Translational Fidelity

Ogle

Ramakrishnan

2005

Annu. Rev. Biochem.

548

538

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■ Abstract The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75Å away. The ribosome recognizes the geometry of codonanticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting. CONTENTS

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Section: Cryo-electron Microscopy Structures Of the Ribosomementioning

confidence: 99%

Structural Insights Into Translational Fidelity

Ogle

Ramakrishnan

2005

Annu. Rev. Biochem.

548

538

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“…The free S Mediator particles all existed in a characteristic compact conformation that was distinctly different from the appearance of free S. cerevisiae pol II in projection (17), which allowed for manual selection of 950 S Mediator particles and calculation of an initial density in EMAN (18). The generated model was used together with the density of pol II in a supervised classification scheme (19) to sort the free S Mediator particles into a homogeneous group before further processing. To validate the classification, we repeated the procedure using our final S Mediator density, which resulted in matching only 450 of 2,400 particles in the S Mediator population to pol II.…”

Section: Electron Microscopy Of the S Pombe Mediator And Pol II Holomentioning

confidence: 99%

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

Elmlund

Baraznenok

Lindahl

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

190

168

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electron microscopy ͉ transcription ͉ protein structure ͉ yeast ͉ Schizosaccharomyces pombe T he Mediator complex acts as an interface between genespecific regulatory proteins and the basal RNA polymerase II (pol II) transcription machinery (1). Mediator functions as a key regulator of pol II-dependent genes in Saccharomyces cerevisiae (2), and depletion of human Mediator from nuclear extracts abolishes transcription by pol II (3). The C-terminal domain of pol II (CTD) has an important role for the Mediator function (4, 5), and no fewer than nine SRB genes, encoding for Mediator subunits, were originally identified in a screen for mutants that suppress the cold-sensitive phenotype of a CTD truncation mutant (6). In S. cerevisiae and Schizosaccharomyces pombe, the Mediator complex interacts directly with the unphosphorylated CTD and forms a holoenzyme (5). Based on shape analysis of the low-resolution projection maps, the S. cerevisiae Mediator structure has been divided into three compact and visually distinguishable modules: head, middle, and tail domains of approximately equal mass (7).The subunit composition of S. cerevisiae Mediator has been studied in detail, and 21 proteins are bona fide members of the core Mediator complex (1,8,9). In addition, a subgroup of Srb proteins, Med12͞Srb8, Med13͞Srb9, Cdk8 (cyclin-dependent kinase 8)͞Srb10, and CycC͞Srb11, forms a specific module (the Cdk8 module) that is variably present in Mediator preparations (10, 11). The smaller, core Mediator (S Mediator) lacking the Cdk8 module has a stimulatory effect on basal transcription in vitro (5, 12). The larger form of Mediator (L Mediator), containing the Cdk8 module, instead represses basal transcription in vitro, and genetic analysis also indicates that the Cdk8 module is involved in the negative regulation of genes in vivo (13).The Cdk8 module influences pol II interactions with Mediator, and only S Mediator can interact with pol II and form a holoenzyme complex (11). The molecular mechanism by which the Cdk8 module negatively regulates pol II interactions has not been clarified, but it has been hypothesized that the negative effect of the Cdk8 module on eukaryotic transcription is caused by Cdk8-dependent phosphorylation of CTD. The hyperphosphorylated form of CTD would bind less tightly to Mediator, which may result in dissociation of pol II from the holoenzyme complex (14, 15).Here, we use the S. pombe system to investigate the molecular basis for the distinct functional properties of S and L Mediator. We find that the Cdk8 module binds to the pol II-binding cleft of Mediator, where it sterically blocks interactions with the polymerase. In contrast to earlier assumptions, the Cdk8 kinase activity is dispensable for negative regulation of pol II interactions with Mediator. It should be noted that the structure and function of Mediator appears conserved in fungi and metazoan cells, and to simplify comparisons with other experimental systems, throughout this study we use the recently proposed unifying Mediator nomenclature ...

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“…Structural studies of tRNA (2)(3)(4)(5), release factors (6 -8), and ribosome recycling factor (9,10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).…”

mentioning

confidence: 99%

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a presteady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.The ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2-5), release factors (6 -8), and ribosome recycling factor (9, 10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).What is the specific nature of these molecular interactions, and what do they suggest about the role of H69 in ribosome function? H69 contacts incoming tRNA in both the pre-and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding pheno...

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Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process

Cited by 286 publications

References 63 publications

Structural Insights Into Translational Fidelity

Structural Insights Into Translational Fidelity

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

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