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Chapter 21 : Modern Site-Directed Cross-Linking Approaches: Implication for Ribosome Structure and Functions

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Abstract:

This chapter reviews modern trends in the development of cross-linking approaches and their implication for studies of rRNA folding in the ribosome and the organization of ribosomal functional centers. It illustrates the results emanating from the ribosomal cross-linking studies with data taken from the authors' work on the investigation of the environment of mRNA and 5S rRNA in the ribosome. The application of a combination of short-range and long-range crosslinking reagents has proved to be a very fruitful strategy for investigation of the topography of RNA and proteins in the ribosome. A section briefly describes the photo-reactive compounds used in most cross-linking experiments with ribosomes. Trifluoromethylaryldiazirines (TFMAD)-containing photoreagents can be used for scanning very fast conformational changes within the ribosome structure. The data reviewed in the chapter show that site-specific photo-cross-linking is a powerful tool for the identification of interacting elements of the translational machinery and for the characterization of functional centers in the ribosome. Furthermore, it has provided invaluable information concerning constraints on ribosome structure which have already been used in the interpretation of crystallographic and electron microscopy data.

Citation: Bogdanov A, Sergiev P, Lavrik I, Spanchenko O, Leonov A, Dontsova O. 2000. Modern Site-Directed Cross-Linking Approaches: Implication for Ribosome Structure and Functions, p 245-255. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch21

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Figure 1

Examples of photoreagents used in ribosome studies. (A) Zero-length reagents. (B) Derivatization of modified uridine residues in the RNA chain with TFMAD.

Citation: Bogdanov A, Sergiev P, Lavrik I, Spanchenko O, Leonov A, Dontsova O. 2000. Modern Site-Directed Cross-Linking Approaches: Implication for Ribosome Structure and Functions, p 245-255. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch21
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Image of Figure 2.
Figure 2.

Summary of the cross-linking data on the environment of mRNA in the ribosome. Secondary-structural elements cross-linked to the mRNA are shown. The solid line in the middle corresponds to the mRNA. The position of the SD sequence is indicated by italic letters. Nucleotides within the variable spacer 59 to the AUG P-site codon are denoted by Ns. The mRNA positions 39 to the AUG P-site codon are shown by numbers. Position-specific cross-link sites are shown by arrows and boldface.

Citation: Bogdanov A, Sergiev P, Lavrik I, Spanchenko O, Leonov A, Dontsova O. 2000. Modern Site-Directed Cross-Linking Approaches: Implication for Ribosome Structure and Functions, p 245-255. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch21
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Figure 3

A network of interactions between the 5S rRNA and domains II and V of 23S rRNA established by cross-linking data. I. Cross-links formed with 4-thioU randomly incorporated into the 5S rRNA ( ). II. Cross-links formed by the diazirine derivative of 5-methyleneaminouridine ( ). III. Crosslink formed by 4-thiouridine attached to residue 1045 of the 23S rRNA ( ). IV. Cross-links formed by APAB, attached to a sequence within the 5S rRNA ( ). The cross-linked rRNA regions are shown as boxes.

Citation: Bogdanov A, Sergiev P, Lavrik I, Spanchenko O, Leonov A, Dontsova O. 2000. Modern Site-Directed Cross-Linking Approaches: Implication for Ribosome Structure and Functions, p 245-255. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch21
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