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Chapter 19 : Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants

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Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, Page 1 of 2

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Abstract:

The recent development of new genetic systems for the construction of rRNA and ribosomal protein mutants in has facilitated both structural and functional analyses of these molecules. Mutations in ribosomal proteins resulting in antibiotic resistance or alterations in the accuracy of translation can now be interpreted in structural terms while the consequences for ribosome structure of alterations in rRNA have been visualized directly for the first time by cryo-electron-microscopic techniques. This chapter reviews the recent developments that the author's have made in this area. They describe some of the recent applications of this strain and its potential for further analysis of rRNA function. The acceptance of the base-pairing model of enhancer action has led to the conclusion that all downstream box (DB) elements enhance translation by the same mechanism and has prompted the identification of DB elements by 16S rRNA sequence complementarity alone. Dihydrouridine (D) is one of the most common posttranscriptional modifications found in bacterial and eukaryal tRNAs, where it is believed to allow conformational flexibility in the loop regions of tRNAs that are involved in tertiary interactions. The development of an strain that permits the expression of pure populations of mutant rRNAs has greatly facilitated genetic, functional, and structural analyses of rRNA.

Citation: O'Connor M, Bayfield M, Gregory S, Lee W, Lodmell J, Mankad A, Thompson J, Vila-Sanjurjo A, Squires C, Dahlberg A. 2000. Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, p 217-227. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch19

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Figures

Image of Figure 1
Figure 1

Secondary structure of 16S rRNA ( ) showing the locations of bases involved in kasugamycin resistance, the 912-888 switch, and proposed sites of mRNA-rRNA interaction.

Citation: O'Connor M, Bayfield M, Gregory S, Lee W, Lodmell J, Mankad A, Thompson J, Vila-Sanjurjo A, Squires C, Dahlberg A. 2000. Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, p 217-227. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch19
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Image of Figure 2
Figure 2

Secondary structures of wild-type and mutant anti-DB regions of helix 44 of 16S rRNA.

Citation: O'Connor M, Bayfield M, Gregory S, Lee W, Lodmell J, Mankad A, Thompson J, Vila-Sanjurjo A, Squires C, Dahlberg A. 2000. Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, p 217-227. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch19
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Image of Figure 3
Figure 3

Secondary structures of the wild-type 460 stem-loop from (left) and (middle) 16S rRNAs and a mutant in which all 8 bp of the stem-loop are reversed (right). The sequences of the original epsilon element from T7 gene 10 ( ), an optimized epsilon element ( ), and the epsilon containing regulatory region of the mRNA ( ) are shown beneath.

Citation: O'Connor M, Bayfield M, Gregory S, Lee W, Lodmell J, Mankad A, Thompson J, Vila-Sanjurjo A, Squires C, Dahlberg A. 2000. Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, p 217-227. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch19
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Image of Figure 4
Figure 4

Two conformational states of helix 27 16S rRNA. (a) 912-885 conformation, with E loop shown in box. (b) 912-888 restrictive conformation, with disrupted E loop. Destabilization of the E loop could trigger the switch between the and restrictive structures. The bases directly involved in the triplet switch are shown in boldface.

Citation: O'Connor M, Bayfield M, Gregory S, Lee W, Lodmell J, Mankad A, Thompson J, Vila-Sanjurjo A, Squires C, Dahlberg A. 2000. Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, p 217-227. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch19
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Image of Figure 5
Figure 5

Effects of mutations in the P loop of 23S rRNA on reactivity to base-specific chemical probes. The positions marked by solid dots exhibit novel or enhanced reactivity in ribosomes bearing mutations at the universally conserved nucleotide G2251 or G2252. The locations of the P and A loops, which interact with P-site- and A-site-bound tRNAs, respectively, are indicated.

Citation: O'Connor M, Bayfield M, Gregory S, Lee W, Lodmell J, Mankad A, Thompson J, Vila-Sanjurjo A, Squires C, Dahlberg A. 2000. Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, p 217-227. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch19
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Image of Figure 6
Figure 6

Effects of erythromycin resistance mutations in ribosomal proteins L22 and L4 ( and , respectively) on the reactivity of 23S rRNA to base-specific chemical probes. Newly reactive positions are indicated by solid dots. The and mutations produce distinct patterns of reactivity across several secondary structure domains.

Citation: O'Connor M, Bayfield M, Gregory S, Lee W, Lodmell J, Mankad A, Thompson J, Vila-Sanjurjo A, Squires C, Dahlberg A. 2000. Probing Ribosomal Structure and Function: Analyses with rRNA and Protein Mutants, p 217-227. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch19
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