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Chapter 37 : Structures and Properties of Ribotoxins

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Abstract:

Ribosome-inactivating proteins (RIPs) are protein toxins produced by organisms ranging from bacteria to plants which specifically damage eukaryotic and prokaryotic ribosomes, rendering them unable to bind elongation factors, and consequently interfering with the elongation steps in translation. RIPs from higher plants can be classified into two categories according to their structures, namely, type I and type II RIPs. Fungal ribotoxins block protein synthesis by inhibiting both the elongation factor 1 (EF-1)- or EF-Tu-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF-2 or EF-G to ribosomes. An understanding of the biology of mitogillin and related fungal ribotoxins at the molecular level has become increasingly important because of their potential application as a component of immunotoxins. Mitogillin and related ribotoxins are known to have amino acid sequence similarity to T1-like ribonucleases, with a unique specificity of interaction with the ribosome causing a single ribonucleolytic cleavage in the large-subunit rRNA. Studies have indicated that the similarities and differences detected in amino acid sequence comparison of ribotoxins and a large family of other guanyl ribonucleases may represent domains or residues essential to ribonucleolytic activity and specificity. The chapter finally focuses on ribosomal recognition elements in mitogillin.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37

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Figures

Image of Figure 1
Figure 1

Nonspecific ribonucleolytic activity of mitogillin and its variants on poly(I). (a) Results were plotted as percent of poly(I) degradation versus protein concentration. (b) Results were plotted as percent of poly(I) degradation versus time when 3 μM mitogillin or a variant was used.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
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Image of Figure 2
Figure 2

Specific ribonucleolytic activity (in vitro fragment release) of mitogillin and its variants. Lane 1, mitogillin; lane 2, no toxin; lane 3, His49Tyr mutant; lane 4, Glu95Lys mutant; lane 5, Arg120Lys mutant; lane 6, His136Tyr mutant. The positions of 28S rRNA, 18S rRNA, and the fragment are indicated.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
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Figure 3

Structural comparison of restrictocin and ribonuclease T1. The secondary structures of the proteins are labeled: 1 to 7, -sheets 1 to 7; L1 to L6, loops 1 to 6; H1 and H2, -helixes 1 and 2. The positions of the catalytically important residues H, E, R, and H of restrictocin and the corresponding residues H, E, R, and H of ribonuclease T1 are also indicated. Note the absence of the B1-L1-B2, L3, and L4 domains of mitogillin in ribonuclease T1. The coordinates of restrictocin and ribonuclease T1 are taken from the Protein Data Bank files 1AQZ ( ) and 1RNT ( ), respectively.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
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Figure 4

Mutagenesis of mitogillin. The sites of deletions are indicated. Also shown are the secondary structures of mitogillin: 1 to 7, -sheets 1 to 7; L1 to L6, loops 1 to 6; H1 and H2, alpha helixes 1 and 2.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
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Image of Figure 5
Figure 5

(A) Specific ribonucleolytic activity (in vitro fragment release) of mitogillin and its variants. The positions of 28S rRNA, 18 S rRNA, and the fragment are indicated. (B) Synthetic -sarcin loop cleavage assay. The positions of the 35- mer, 21-mer, and 14-mer are also indicated. Lanes 1, mitogillin; lanes 2, no toxin; lanes 3, ΔK–K mutant; lanes 4, ΔK– D mutant; lanes 5, ΔK–L mutant; lanes 6, ΔK–S mutant; lanes 7, ΔD–K mutant; lanes 8, ΔG–I mutant; lanes 9, ΔK–I mutant; lanes 10,ΔR–Q mutant; lanes 11, ΔN–K mutant; and lanes 12, ΔK–K mutant.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
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Figure 6

Nonspecific ribonucleolytic activity of mitogillin and its mutants on poly(I) substrate. The results were plotted as percent of poly(I) degradation versus time when 3 μM of mitogillin or a mutant protein was used.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
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Image of Figure 7
Figure 7

Hydrogen-bonding between B1-L1-B2 and B6-L6-B7 domains of restrictocin. L1 residues 11 to 16 are fully exposed to solvent and are consequently missing in the atomic model. Hydrogen bonds are denoted by dashed lines. Amino acid residues not relevant in forming hydrogen bonds are omitted for clarity.

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
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Tables

Generic image for table
Table 1

Representative RIPs from various sources

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
Generic image for table
Table 2

Production of mutant mitogillin proteins

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
Generic image for table
Table 3

Homologous motifs found in ribotoxins and in ribosomal protein S12 from a variety of sources

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
Generic image for table
Table 4

Homologous motifs found in mitogillin and in translation elongation factors

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37
Generic image for table
Table 5

Comparison of the ribonucleolytic activities (initial rate of cleavage) of mitogillin, mutant mitogillins, and ribonuclease T1 on poly(I) homopolymer

Citation: Kao R, Davies J. 2000. Structures and Properties of Ribotoxins, p 451-460. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch37

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