Cryo-Electron Microscopy of the Translational Apparatus: Experimental Evidence for the Paths of mRNA, tRNA, and the Polypeptide Chain

Joachim Frank; Robert A. Grassucci; Amy Heagle; Christian M. T. Spahn; Pawel Penczek; Rajendra K. Agrawal

doi:10.1128/9781555818142.ch5

Chapter 5 : Cryo-Electron Microscopy of the Translational Apparatus: Experimental Evidence for the Paths of mRNA, tRNA, and the Polypeptide Chain

Authors: Joachim Frank¹, Robert A. Grassucci¹, Amy Heagle¹, Christian M. T. Spahn¹, Pawel Penczek², Rajendra K. Agrawal²

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Affiliations: 1: Health Research, Inc., at the Wadsworth Center and Howard Hughes Medical Institute, Empire State Plaza, Albany, NY 12201-0509; 2: Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509

Content Type: Monograph

Publication Year: 2000

Category: Microbial Genetics and Molecular Biology

Book DOI: 10.1128/9781555818142

Chapter DOI: 10.1128/9781555818142.ch5

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Abstract:

As a process that brings together, in close proximity, two linear structures of considerable length (mRNA and the nascent polypeptide chain) and large protein factors (EF-G and aminoacyl-tRNA·EF-Tu·GTP ternary complex), protein synthesis poses a logistic problem of traffic control: how to guarantee uninterrupted, high-precision performance without steric interference and entanglement of the various ligands. Since cryo-electron microscopy (cryo-EM) visualization provided the first detailed three-dimensional (3-D) images of the ribosome, much work has gone into the mapping of tRNA and elongation factors bound to the ribosome at various stages of the elongation cycle. A recent study of a 70S ribosome carrying a genetically inserted tRNA-like RNA fragment furnished a higher-resolution (17-Å) map of the vacant ribosome, and the use of this new map in the subtraction produced a linear mass distribution covering the platform side segment of the mRNA path, as well as a mass hovering just at the entrance of the 30S subunit channel. tRNA bound to the ribosome has been directly visualized by 3-D cryo-EM in various tRNA-ribosome complexes. Visualization of the ribosome-bound tRNA is still a challenging task because the smallest dimension of the molecule is on the order of the resolution of cryo-EM and the occupancy of some tRNA binding states is intrinsically low.

Citation: Frank J, Grassucci R, Heagle A, Spahn C, Penczek P, Agrawal R. 2000. Cryo-Electron Microscopy of the Translational Apparatus: Experimental Evidence for the Paths of mRNA, tRNA, and the Polypeptide Chain, p 45-51. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch5

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Cryo-Electron Microscopy of the Translational Apparatus: Experimental Evidence for the Paths of mRNA, tRNA, and the Polypeptide Chain

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/content/10.1128/9781555818142.chap5.fig5-1

Figure 1

(Top left) Uncut 15-Å map of the fMet-tRNAf Met•ribosome complex ( Malhotra et al., 1998 ) in the view selected for the middle left and bottom left panels. Landmarks, 50S subunit: CP, central protuberance; L1, L1 protein 30S subunit; pt, platform; sp, spur. (Middle left) Experimental evidence for the path of mRNA, obtained from two difference maps, superimposed on the 15-Å map of the ribosome that was cut open to reveal the intersubunit space and the relevant plane of mRNA movement. The color code is as follows: (i) Green indicates the result of subtraction of the mRNA-free control from the fMet-tRNA•ribosome complex map, displayed with reduced threshold. As a result, the difference mass corresponding to the P-site tRNA is enlarged compared to its appearance in the study of Malhotra et al. (1998), but it also extends along a curved path (MF) toward the left, into the space between the platform and the head. Another globular mass probably also related to mRNA occurs in the channel. Its position is slightly above the cutting plane, toward the viewer. The other peaks of this difference map are probably related to conformational changes between the naked ribosome and the tRNA-bound ribosome. (ii) Brown indicates the result of subtraction of the vacant-ribosome map from the map of the polysome. The difference mass follows a curved arc (polysome) through the 30S channel, partially overlapping (dashed outline) and complementing the path of the green MF-mRNA-related difference mass. The other difference peaks are related to low-occupancy A-site tRNA (A), E-site tRNA (E), and E2-site tRNA (E2) and to conformational changes. (Bottom left) Path of mRNA (dashed line) inferred from the experimental difference maps, along with a model of P-site tRNA that was inserted in the position found in the study of Malhotra et al. (1998). (Top right) Uncut 15-Å map of the fMet-tRNAf Met•ribosome complex ( Malhotra et al., 1998 ) in the view selected for the middle right and bottom right panels. h, head of the 30S subunit. (Middle right) Difference mass observed in the tunnel of the ribosome when the map of the polysome is compared with that of the empty ribosome. The difference mass has been overlaid on the 15-Å map cut open along the plane of the tunnel. The mass attributed to partially folded polypeptide chain (PPC) occurs in the widened midsection of the tunnel. The other difference peaks relate to the presence of tRNA at the A site (A), P-site tRNA (P), E-site tRNA (E), and the exit site of the tunnel (EX1). (Note that the side branch of the tunnel, ending in the second tunnel exit side EX2, is not visible in this orientation; cf. Frank et al., 1995 .) (Bottom right) Same view of 15-Å map, with X-ray structure of P-site tRNA in fitted position and probable path of polypeptide chain indicated (dashed line).

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Figure 1

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Figure 2

(Top) Schematic diagram of the movements of tRNA and elongation factors during the elongation cycle of protein synthesis. The ribosome map and the 3-D positions of the ligand molecules are based on several cryo-EM visualizations (for an explanation of panels (i) through (iv), see the text). (Bottom) Examples of the fitting of the X-ray structure of tRNA to experimental difference masses: E2 site (above) and P site (below). Density belonging to the neighboring tRNA is marked by “#” in both maps.

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Figure 2

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