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Chapter 10 : How Ribosomal Proteins and rRNA Recognize One Another

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Abstract:

The assembly and structural integrity of the ribosome are guaranteed by specific interactions among its component proteins and RNAs. In and other bacteria, many of the proteins that associate with rRNA also regulate the translation of ribosomal protein operons via interactions with the corresponding mRNAs. In all likelihood, these features define the specific three-dimensional surfaces that are recognized by the ribosomal proteins. The results discussed in this chapter focus on the studies of the interactions between ribosomal proteins S8 and L1 and their binding sites in the 16S and 23S rRNAs, respectively, which take place during ribosomal subunit assembly. The results demonstrated that the difference in the affinity of S8 for the two sites can in fact be attributed to the bulged bases. The conservation of nucleotides within the conserved core of the S8 binding site—whether in rRNA or in mRNA—is remarkable, and from evidence to be presented, it is clearly related to the capacity of both RNAs to associate with S8. Site-directed-mutagenesis experiments have shown that the identities of most of the nucleotides in the conserved core (nucleotides 595 to 598 and 640 to 644) are critical for optimal S8-RNA interaction, whereas almost all of the base pairs in the duplex segments can be replaced with only minimal effects on protein binding as long as the helical elements are maintained.

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10

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Figures

Image of Figure 1
Figure 1

Binding site for protein S8. (Left) Structures of the binding site for ribosomal protein S8 in 16S rRNA (left) and mRNA (right) from . (Right) Position of the S8 binding site in the secondary structure of the 16S rRNA. Helices 21 and 25 are denoted as H21 and H25, respectively.

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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Image of Figure 2
Figure 2

Phylogenetic conservation and mutagenesis in the binding site for protein S8. (Left) Conservation of nucleotides in bacterial and chloroplast 16S rRNAs; boldface, <95%; shadow, 90 to 95%; outline, 80 to 90%; hatched, constrained to A or U in over 93% of prokaryotic 16S rRNAs. (Right) Nucleotides which, when mutated, sharply decrease S8-RNA affinity (boldface).

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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Image of Figure 3
Figure 3

Base triples in the binding site for protein S8. (a) A595•(A596-U644). (b) U641•(G597-C643).

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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Image of Figure 4
Figure 4

Effects of nucleotide analogue substitutions on S8-RNA interaction. Abbreviations are: r, ribo; d, 2′-deoxy; 2′OMe, 2′-methyl; sU, 4-thiouridine; Pu, purine; I, inosine; 2APu, 2- aminopurine; 7deaza, 7-deaza.

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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Image of Figure 5
Figure 5

Structure of protein S8 showing amino acids implicated in RNA binding. Over 40 single-site mutants of protein S8 were isolated in a genetic screen designed to identify variants defective in RNA binding ( ). Altered amino acids, depicted in ball-and-stick mode, are displayed on the backbone structure of protein S8 from according to the crystallographic structure determined by . The effects of the amino acid replacements on S8-RNA interaction are indicated as follows: red, strongly deleterious; red-orange, mildly deleterious; orange, no effect; yellow, identified in screen but not yet tested. Gln56 and Tyr86, in magenta, correspond to Lys55 and Tyr85 in S8 (see the text).

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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Figure 6

Binding site for protein L1. (Left) Structures of the binding site for ribosomal protein L1 in 23S rRNA (top) and L1-L11 mRNA (bottom) from . (Right) Position of the L1 binding site in the secondary structure of the 3′ half of the 23S rRNA. Helices 76, 77, and 78 are denoted as H76, H77, and H78, respectively.

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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Image of Figure 7
Figure 7

Phylogenetic conservation and mutagenesis in the binding site for protein L1. (Left) Conservation of nucleotides in bacterial and chloroplast 16S rRNAs; boldface, <95%; shadow, 90 to 95%; outline, 80 to 90%. (Right) Nucleotides which, when mutated, sharply decrease L1-RNA affinity (boldface).

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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Image of Figure 8
Figure 8

Modification-interference analysis of L1 binding site partially substituted with rUS. (a) Autoradiogram of rUS-substituted RNA after RNase U, alkali, and iodine treatment, followed by separation on a 15% polyacrylamide gel. Lane U, RNase U digestion; lane OH, partial alkaline hydrolysis; lane I2 total, iodine cleavage of unfractionated RNA; lane I2 selected, iodine cleavage of L1-bound RNA. (b) Relative intensities of bands corresponding to individual U residues after iodine cleavage, as measured by phosphorimaging. A value near 1.0 indicates no enrichment of rU over rUS in the bound fraction. Reduced values indicate a preference for the unmodified nucleotide.

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10
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References

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Tables

Generic image for table
Table 1

Relative affinities of truncated variants of protein S8 for the S8 binding site

Citation: Zimmermann R, Alimov I, Uma K, Wu H, Wower I, Drygin D, Dong P, Jiang L, Nikonowicz E. 2000. How Ribosomal Proteins and rRNA Recognize One Another, p 93-104. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch10

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