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Chapter 15 : Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli

Authors: Margaret S. Vanloock1, Thomas R. Easterwood2, Stephen C. Harvey3, Arun Malhotra4, Rajendra K. Agrawal5, Pawel Penczek6, David A. Case7, Joachim Frank8
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Affiliations: 1: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005; 2: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005; 3: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005; 4: Wadsworth Center, P.O. Box 509, Albany, NY 12201-0509; 5: Wadsworth Center, P.O. Box 509, Albany, NY 12201-0509; 6: Wadsworth Center, P.O. Box 509, Albany, NY 12201-0509; 7: Department of Molecular Biology, The Scripps Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037; 8: Howard Hughes Medical Institute, P.O. Box 509, Albany, NY 12201-0509
Content Type: Monograph
Publication Year: 2000

Category: Microbial Genetics and Molecular Biology

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Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli, Page 1 of 2

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Abstract:

Recently, the everimproving cryo-electron microscopy maps of the 70S ribosome have increased the overall accuracy of the modeling studies. This chapter talks about three important structure-function relationships. First, it briefly reviews the recently published model of interactions between the 16S rRNA decoding site and the mRNAtRNA complex. Second, it discusses the functional implications of the placement of that model in the electron density map. Finally, it identifies regions that the authors believe compose the ‘‘gate’’ that closes to form the channel in which the mRNA is located. The second-generation models incorporate atomic-resolution data for those components whose structures have been determined by crystallography or nuclear magnetic resonance (NMR), and for selected components the authors have modeled at atomic detail because of their functional significance. The authors therefore predict that the density in the channel will rise when tRNA is bound at the A site. Another interesting aspect of this model is that the axis of the decoding-site RNA lies perpendicular to the long axis of the 30S ribosomal subunit. Cross-linking studies between mRNA and the 16S rRNA also suggest that these two regions of the 16S rRNA are close together in the ribosomal subunit. Multiscale modeling permits investigation of structure-function relationships at atomic detail within the low-resolution framework of those regions that are less well defined and less functionally significant.

Citation: Vanloock M, Easterwood T, Harvey S, Malhotra A, Agrawal R, Penczek P, Case D, Frank J. 2000. Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli, p 165-172. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch15

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Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli
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Citation: Vanloock M, Easterwood T, Harvey S, Malhotra A, Agrawal R, Penczek P, Case D, Frank J. 2000. Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli, p 165-172. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch15
/content/10.1128/9781555818142.chap15.fig15-1
Figure 1

Secondary-structure diagram of the 16S rRNA from E. coli ( ). The decoding site region and helices 18 and 34 have been labeled.

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Figure 1

Secondary-structure diagram of the 16S rRNA from E. coli ( ). The decoding site region and helices 18 and 34 have been labeled.

Citation: Vanloock M, Easterwood T, Harvey S, Malhotra A, Agrawal R, Penczek P, Case D, Frank J. 2000. Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli, p 165-172. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch15
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Figure 2

Ribbon diagram ( ) of the 16S rRNA decoding- site model ( ) with the mRNA-tRNA complex bound in the major groove of the 16S rRNA. A1492 and A1493 (cyan) form sequence-independent interactions with the Asite codon of the mRNA (green). C1400 (red) stacks directly beneath the wobble base of the P-site tRNA (yellow), as was indicated by a UV-induced cross-link between these two residues ( ). The A-site tRNA is shown in blue.

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10.1128/9781555818142/fig15-2.gif
Image of Figure 2
Figure 2

Ribbon diagram ( ) of the 16S rRNA decoding- site model ( ) with the mRNA-tRNA complex bound in the major groove of the 16S rRNA. A1492 and A1493 (cyan) form sequence-independent interactions with the Asite codon of the mRNA (green). C1400 (red) stacks directly beneath the wobble base of the P-site tRNA (yellow), as was indicated by a UV-induced cross-link between these two residues ( ). The A-site tRNA is shown in blue.

Citation: Vanloock M, Easterwood T, Harvey S, Malhotra A, Agrawal R, Penczek P, Case D, Frank J. 2000. Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli, p 165-172. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch15
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Figure 3

Ribbon image showing the position of the 16S rRNA decoding-site model ( ) docked into the 15-Å cryo-electron microscopy map of the intact 70S ribosome ( ). The mRNA (green) lies in the center of the channel that is surrounded by the decoding-site RNA (red). This cryo-electron microscopy reconstruction has an fMet-tRNAfMet (yellow) bound to the P site and no tRNA bound to the A site. However, the A-site tRNA (blue) from the decoding-site model was included in this docking exercise. The P-site tRNA (yellow) is also visible in this image. The reason for this is threefold: first, the threshold of the electron microscopy map is high, thus eliminating regions of low electron density; second, the P site is not fully occupied by fMet-tRNAfMet; and third, there is motion in the acceptor stem. All three of these factors together decrease the electron density in the region of the P-site tRNA, thus making it visible in this figure.

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Image of Figure 3
Figure 3

Ribbon image showing the position of the 16S rRNA decoding-site model ( ) docked into the 15-Å cryo-electron microscopy map of the intact 70S ribosome ( ). The mRNA (green) lies in the center of the channel that is surrounded by the decoding-site RNA (red). This cryo-electron microscopy reconstruction has an fMet-tRNAfMet (yellow) bound to the P site and no tRNA bound to the A site. However, the A-site tRNA (blue) from the decoding-site model was included in this docking exercise. The P-site tRNA (yellow) is also visible in this image. The reason for this is threefold: first, the threshold of the electron microscopy map is high, thus eliminating regions of low electron density; second, the P site is not fully occupied by fMet-tRNAfMet; and third, there is motion in the acceptor stem. All three of these factors together decrease the electron density in the region of the P-site tRNA, thus making it visible in this figure.

Citation: Vanloock M, Easterwood T, Harvey S, Malhotra A, Agrawal R, Penczek P, Case D, Frank J. 2000. Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli, p 165-172. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch15
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Figure 4

The mRNA within the channel, viewed from the direction of the large subunit. At this density threshold, the mRNA in the P site is buried in the density, reflecting the well-defined structure of the tRNA, mRNA, and 16S RNA in the P site. A lack of tRNA in the A site produces a dynamic, poorly defined structure for the A-site mRNA-rRNA complex. This is reflected by the lower density in the A-site part of the channel and the visibility of the A-site mRNA in this model.

10.1128/9781555818142/fig15-4_thmb.gif
10.1128/9781555818142/fig15-4.gif
Image of Figure 4
Figure 4

The mRNA within the channel, viewed from the direction of the large subunit. At this density threshold, the mRNA in the P site is buried in the density, reflecting the well-defined structure of the tRNA, mRNA, and 16S RNA in the P site. A lack of tRNA in the A site produces a dynamic, poorly defined structure for the A-site mRNA-rRNA complex. This is reflected by the lower density in the A-site part of the channel and the visibility of the A-site mRNA in this model.

Citation: Vanloock M, Easterwood T, Harvey S, Malhotra A, Agrawal R, Penczek P, Case D, Frank J. 2000. Functional Interpretation of the Cryo-Electron Microscopy Map of the 30S Ribosomal Subunit from Escherichia coli, p 165-172. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch15
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