Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy

Valle, Mikel; Zavialov, Andrey V.; Li, Wen; Stagg, Scott M.; Sengupta, Jayati; Nielsen, Rasmus Gaardskær; Nissen, Poul; Harvey, Stephen C.; Ehrenberg, Måns; Frank, Joachim

doi:10.1038/nsb1003

Cited by 311 publications

(266 citation statements)

References 42 publications

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“…2a). This movement is in a direction similar to those observed previously upon binding of elongation factors (23,24) and release factor 2 (6). The mass of density that appears (green in Fig.…”

Section: Resultssupporting

confidence: 70%

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Agrawal

Sharma

Kiel

et al. 2004

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

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148

170

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After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosomerecycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by Ϸ13 Å) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA. R ibosomes are responsible for translating genetic information carried by mRNAs into specific sequences of amino acids. Translation on the ribosome comprises of four main steps: (i) initiation, (ii) elongation, (iii) termination, and (iv) recycling. Recent advancements in structural studies of the translational machinery have helped elucidate binding positions and functions of various translational factors involved in various stages of initiation (1, 2), elongation (3, 4), and termination (5-7). The fourth step of translation requires binding of a dedicated protein factor, the ribosome-recycling factor (RRF), which in conjunction with elongation factor G (EF-G) helps removing the mRNA and last deacylated tRNA from the ribosome (see ref. 8).Atomic structures of RRF determined from five different species, including Escherichia coli, show that it is comprised of two structural domains: domain I, consisting of three long ␣-helix bundles, and the smaller domain II, which is an ␣͞␤ motif (9-13). Different orientations of domain II in these structures have been attributed to interdomain flexibility, which is thought to be necessary for RRF to function on the ribosome (12).The overall match in dimensions between RRF and tRNA (9) prompted the proposal of structural and functional molecular mimicry between the two molecules (9). In a recent study (14) using the hydroxyl radical probing (HRP) method, the orientation of RRF on the ribosome was derived. This study did not support the idea of direct molecular mimicry of tRNA by RRF, because the derived binding position of RRF was quite different from that one would expect based on structural mimicry. The inferred bin...

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Section: Resultssupporting

confidence: 70%

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Agrawal

Sharma

Kiel

et al. 2004

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

Self Cite

148

170

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“…Cryo-electron microscopic structures of these stabilizing contacts of the ternary complex with the 50S subunit of the ribosome, particularly with the sarcin-ricin loop (SRL) and the GTPase-associated center (GAC) are shown in supporting information (SI) Fig. 5 (5,(10)(11)(12).…”

mentioning

confidence: 99%

“…GTPase activation takes place after additional stabilizing contacts are formed between the ribosome and the ternary complex (6,8). In the initial selection of aa-tRNA, the ternary complex binding induces structural changes in the ribosome (''induced-fit'') (5,9) in which the 30S subunit of the ribosome changes its structure and forms additional stabilizing contacts with the incoming tRNA after the codon recognition (9). In case of one base-mismatch out of the 3 bp (near cognate), additional binding contacts are weaker, presumably because the mismatched bases form a less compact structure for the ribosome to ''wrap'' around as it forms additional induced fit contacts.…”

mentioning

confidence: 99%

“…In the past decades, considerable progress has been made in understanding the ingenious mechanisms used by the ribosome to achieve this low error rate (2,(4)(5)(6). The overall selection process consists of initial selection of the ternary complex [aminoacyl-tRNA (aa-tRNA), GTP, and elongation factor Tu (EF-Tu)], GTP hydrolysis in EF-Tu, and then proofreading (6).…”

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confidence: 99%

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The role of fluctuations in tRNA selection by the ribosome

Lee

Blanchard

Kim

et al. 2007

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

144

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The detailed mechanism of how the ribosome decodes protein sequence information with an abnormally high accuracy, after 40 years of study, remains elusive. A critical element in selecting correct transfer RNA (tRNA) transferring correct amino acid is ''induced fit'' between the ribosome and tRNA. By using singlemolecule methods, the induced fit mechanism is shown to position favorably the correct tRNA after initial codon recognition. We provide evidence that this difference in positioning and thermal fluctuations constitutes the primary mechanism for the initial selection of tRNA. This work demonstrates thermal fluctuations playing a critical role in the substrate selection by an enzyme.ribosome dynamics ͉ ribosome selection ͉ single-molecule FRET T ranslation, protein synthesis by the ribosome, is a vital cellular process involving the two-subunit ribosomal particle, multiple RNAs, and protein cofactors (1, 2). To synthesize a protein molecule with correct sequence, the ribosome has to select correct transfer RNA (tRNA) as dictated by messenger RNA (mRNA). In the decoding site of the ribosome, the proper matching of the 3-nt codon sequence of the mRNA to the anticodon sequence of tRNA occurs at a rate of 20-30 per second with an error rate of 10 Ϫ4 (3).The difference in base-pairing energy between codon and anticodon cannot explain the low error rate of translation (4). In the past decades, considerable progress has been made in understanding the ingenious mechanisms used by the ribosome to achieve this low error rate (2, 4-6). The overall selection process consists of initial selection of the ternary complex [aminoacyl-tRNA (aa-tRNA), GTP, and elongation factor Tu (EF-Tu)], GTP hydrolysis in EF-Tu, and then proofreading (6). The GTP hydrolysis ensures that initial selection is separated from the proofreading by an irreversible reaction. This two-step selection yields overall probability of an error to be the product of the error probabilities of the two selection steps, thus dramatically lowering the overall chance of error (7).The initial selection of aa-tRNA consists of initial binding of the ternary complex on the ribosome, interaction between tRNA and mRNA (the codon recognition), and stabilization of the ternary complex onto the ribosome. GTPase activation takes place after additional stabilizing contacts are formed between the ribosome and the ternary complex (6,8). In the initial selection of aa-tRNA, the ternary complex binding induces structural changes in the ribosome (''induced-fit'') (5, 9) in which the 30S subunit of the ribosome changes its structure and forms additional stabilizing contacts with the incoming tRNA after the codon recognition (9). In case of one base-mismatch out of the 3 bp (near cognate), additional binding contacts are weaker, presumably because the mismatched bases form a less compact structure for the ribosome to ''wrap'' around as it forms additional induced fit contacts. In other words, the tighter binding of cognate tRNA relative to near-cognate tRNA mainly is attributable...

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“…Instead, the scanned micrographs were aligned to one another using high-contrast features so that the same particles could be selected from the averaged micrographs ( Figure 1) using an automated particle picking procedure ( Figure 2) [1]. Provided the number of particles is sufficient, the homogeneity of the sample, the E. coli ribosome-bound kirromycin-stalled aa-tRNA·EF-Tu·GDP ternary complex, should result in a high-resolution 3D reconstruction [2] (see Figure 3). …”

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confidence: 99%

Efficient Strategy for Processing Single Particle Data from a Cryo-EM Exposure Series

Grassucci

Baxter

Barnard

et al. 2005

MAM

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Processing data from a frozen hydrated biological sample is inherently difficult because of the low signal-to-noise ratio of the low electron dose images. This low dose is necessary in order to minimize the damage to the biological sample from exposure to the electron beam. A strategy has been developed to use a higher-signal image generated from the average of a series of low dose images for the majority of the processing sequence while still maintaining the ability to calculate a final reconstruction from the original low-dose data.In order to study the effect of dose on biological samples, a series of four exposures were collected from the same area of a ribosome specimen at 300 kV and 12 electrons/Å 2 for each exposure. The standard approach of extracting, aligning and reconstructing each of the four data sets separately was problematic because of the high noise level of the individual micrographs. Instead, the scanned micrographs were aligned to one another using high-contrast features so that the same particles could be selected from the averaged micrographs (Figure 1) using an automated particle picking procedure (Figure 2) [1]. Provided the number of particles is sufficient, the homogeneity of the sample, the E. coli ribosome-bound kirromycin-stalled aa-tRNA·EF-Tu·GDP ternary complex, should result in a high-resolution 3D reconstruction [2] (see Figure 3).An investigation was done to determine how extensively the high-signal averages could be utilized without degrading the final reconstruction (Figure 4). Along with the increase in signal, using these averages also decreases the computation time when compared to processing each exposure separately.The procedure used to generate a reconstruction consists of 4 major steps. In each of these steps one could potentially take advantage of the averaged images.Step 1 is particle picking, which we have determined works with the averaged micrographs as shown in Figure 2. Step 2 requires aligning and matching the experimental projections to the corresponding reference projections. This can be done with averaged particles, and the resulting output documents can be used to align and determine the projection angles for the single exposure particles.Step 3 uses the output of this step to generate a preliminary 3-D reconstruction. In step 4 the preliminary reconstruction is then used in place of the original reference to iteratively generate a series of refined volumes. A preliminary 3-D reconstruction from the averaged particles can be substituted and used as the starting reference to generate the refined volumes. The resolution of the various reconstructions are then compared to determine at what point use of the average data results in an inferior outcome [3].

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Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy

Cited by 311 publications

References 42 publications

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

The role of fluctuations in tRNA selection by the ribosome

Efficient Strategy for Processing Single Particle Data from a Cryo-EM Exposure Series

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