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A minimized rRNA-binding site for ribosomal protein S4 and its implications for 30S assembly.

Bellur DL, Woodson SA - Nucleic Acids Res. (2009)

Bottom Line: S4 binds the minimal 5WJ RNA containing just the five-helix junction as well or better than with affinity comparable to or better than the 5' domain or native 16S rRNA.Hydroxyl radical footprinting and chemical base modification showed that S4 makes the same interactions with minimal rRNA substrates as with the native 16S rRNA, but the minimal substrates are more pre-organized for binding S4.Together, these results suggest that favorable interactions with S4 offset the energetic penalty for folding the 16S rRNA.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, Baltimore, MD 21218-2685, USA.

ABSTRACT
Primary ribosomal protein S4 is essential for 30S ribosome biogenesis in eubacteria, because it nucleates subunit assembly and helps coordinate assembly with the synthesis of its rRNA and protein components. S4 binds a five-helix junction (5WJ) that bridges the 5' and 3' ends of the 16S 5' domain. To delineate which nucleotides contribute to S4 recognition, sequential deletions of the 16S 5' domain were tested in competitive S4-binding assays based on electrophoretic mobility shifts. S4 binds the minimal 5WJ RNA containing just the five-helix junction as well or better than with affinity comparable to or better than the 5' domain or native 16S rRNA. Internal deletions and point mutations demonstrated that helices 3, 4, 16 and residues at the helix junctions are necessary for S4 binding, while the conserved helix 18 pseudoknot is dispensable. Hydroxyl radical footprinting and chemical base modification showed that S4 makes the same interactions with minimal rRNA substrates as with the native 16S rRNA, but the minimal substrates are more pre-organized for binding S4. Together, these results suggest that favorable interactions with S4 offset the energetic penalty for folding the 16S rRNA.

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Escherichia coli 16S rRNA fragments tested for S4 binding. (a) 16S rRNA; (b) 5′domain; (c) RNA(▵H6-14); (d) RNA(▵H5-14); (e) 5WJ RNA; (f) 5WJ_nt6; (g) 5WJ:BstH17; (h) 5WJ:TthH17; (i) 5WJ▵H3; (j) 5WJ▵H4; (k) 5WJ:H16trunc; (l) 5WJ:H17trunc; and (m) 5WJ:H18trunc. Secondary structures of ▵H6-14 and 5WJ were confirmed by RNase T1 digestion (Figures S1 and S2).
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Figure 1: Escherichia coli 16S rRNA fragments tested for S4 binding. (a) 16S rRNA; (b) 5′domain; (c) RNA(▵H6-14); (d) RNA(▵H5-14); (e) 5WJ RNA; (f) 5WJ_nt6; (g) 5WJ:BstH17; (h) 5WJ:TthH17; (i) 5WJ▵H3; (j) 5WJ▵H4; (k) 5WJ:H16trunc; (l) 5WJ:H17trunc; and (m) 5WJ:H18trunc. Secondary structures of ▵H6-14 and 5WJ were confirmed by RNase T1 digestion (Figures S1 and S2).

Mentions: The binding site for ribosomal protein S4 consists of the five-way junction (5WJ) between helices (H) 3, 4, 16, 17 and 18, which flank the 5′ and 3′ ends of the 16S 5′ domain (10,12,13) (Figure 1a and b). The stable C-terminal domains of S4, which include a winged-helix motif (14), directly contacts the center of the 5WJ in the rRNA (15). Outside of a few base-specific contacts in the 5WJ, S4 predominantly interacts with the rRNA backbone.Figure 1.


A minimized rRNA-binding site for ribosomal protein S4 and its implications for 30S assembly.

Bellur DL, Woodson SA - Nucleic Acids Res. (2009)

Escherichia coli 16S rRNA fragments tested for S4 binding. (a) 16S rRNA; (b) 5′domain; (c) RNA(▵H6-14); (d) RNA(▵H5-14); (e) 5WJ RNA; (f) 5WJ_nt6; (g) 5WJ:BstH17; (h) 5WJ:TthH17; (i) 5WJ▵H3; (j) 5WJ▵H4; (k) 5WJ:H16trunc; (l) 5WJ:H17trunc; and (m) 5WJ:H18trunc. Secondary structures of ▵H6-14 and 5WJ were confirmed by RNase T1 digestion (Figures S1 and S2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665224&req=5

Figure 1: Escherichia coli 16S rRNA fragments tested for S4 binding. (a) 16S rRNA; (b) 5′domain; (c) RNA(▵H6-14); (d) RNA(▵H5-14); (e) 5WJ RNA; (f) 5WJ_nt6; (g) 5WJ:BstH17; (h) 5WJ:TthH17; (i) 5WJ▵H3; (j) 5WJ▵H4; (k) 5WJ:H16trunc; (l) 5WJ:H17trunc; and (m) 5WJ:H18trunc. Secondary structures of ▵H6-14 and 5WJ were confirmed by RNase T1 digestion (Figures S1 and S2).
Mentions: The binding site for ribosomal protein S4 consists of the five-way junction (5WJ) between helices (H) 3, 4, 16, 17 and 18, which flank the 5′ and 3′ ends of the 16S 5′ domain (10,12,13) (Figure 1a and b). The stable C-terminal domains of S4, which include a winged-helix motif (14), directly contacts the center of the 5WJ in the rRNA (15). Outside of a few base-specific contacts in the 5WJ, S4 predominantly interacts with the rRNA backbone.Figure 1.

Bottom Line: S4 binds the minimal 5WJ RNA containing just the five-helix junction as well or better than with affinity comparable to or better than the 5' domain or native 16S rRNA.Hydroxyl radical footprinting and chemical base modification showed that S4 makes the same interactions with minimal rRNA substrates as with the native 16S rRNA, but the minimal substrates are more pre-organized for binding S4.Together, these results suggest that favorable interactions with S4 offset the energetic penalty for folding the 16S rRNA.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, Baltimore, MD 21218-2685, USA.

ABSTRACT
Primary ribosomal protein S4 is essential for 30S ribosome biogenesis in eubacteria, because it nucleates subunit assembly and helps coordinate assembly with the synthesis of its rRNA and protein components. S4 binds a five-helix junction (5WJ) that bridges the 5' and 3' ends of the 16S 5' domain. To delineate which nucleotides contribute to S4 recognition, sequential deletions of the 16S 5' domain were tested in competitive S4-binding assays based on electrophoretic mobility shifts. S4 binds the minimal 5WJ RNA containing just the five-helix junction as well or better than with affinity comparable to or better than the 5' domain or native 16S rRNA. Internal deletions and point mutations demonstrated that helices 3, 4, 16 and residues at the helix junctions are necessary for S4 binding, while the conserved helix 18 pseudoknot is dispensable. Hydroxyl radical footprinting and chemical base modification showed that S4 makes the same interactions with minimal rRNA substrates as with the native 16S rRNA, but the minimal substrates are more pre-organized for binding S4. Together, these results suggest that favorable interactions with S4 offset the energetic penalty for folding the 16S rRNA.

Show MeSH
Related in: MedlinePlus