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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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Dual-label competitive binding assay. (a) Scheme of the competitive binding experiments with two labeled RNAs. Complexes of the 5′ domain were separated on 8% polyacrylamide gels in TBE, while complexes of the 5WJ variants were separated on 6% polyacrylamide gels in TKM2, as described in ‘Material and Methods’ sections. (b) Distribution of S4 between 5′ domain and 5WJ RNAs. The counts in each complex and in free RNA were quantified and used to calculate Krel [Equation (2)]. For the 5WJ RNA, average Krel = 0.6 ± 0.1. The amount of labeled 5WJ complex decreases as unlabeled 5WJ RNA is added because its specific activity decreases. See Table 1 for further data.
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Figure 3: Dual-label competitive binding assay. (a) Scheme of the competitive binding experiments with two labeled RNAs. Complexes of the 5′ domain were separated on 8% polyacrylamide gels in TBE, while complexes of the 5WJ variants were separated on 6% polyacrylamide gels in TKM2, as described in ‘Material and Methods’ sections. (b) Distribution of S4 between 5′ domain and 5WJ RNAs. The counts in each complex and in free RNA were quantified and used to calculate Krel [Equation (2)]. For the 5WJ RNA, average Krel = 0.6 ± 0.1. The amount of labeled 5WJ complex decreases as unlabeled 5WJ RNA is added because its specific activity decreases. See Table 1 for further data.

Mentions: To evaluate whether our results were strongly biased by nonspecific binding of S4 (31), we also performed competitive binding experiments where both RNAs were 32P-labeled (Figure 3a). The advantage of this method is that the protein concentration is lower than the total RNA concentration, minimizing contributions from nonspecific interactions (34). To detect S4 binding to two labeled RNA substrates, an aliquot of each binding reaction was loaded on an 8% TBE gel to resolve the 5′ domain complexes, while another aliquot was loaded on a 6% TKM2 gel to resolve the smaller RNA complexes (Figure 3b). The results of the dual-label competition experiments were in good agreement with the competition assays done with only labeled 5′ domain and excess S4 (Table 1). Thus, we concluded that both sets of competition experiments were likely reporting the relative stabilities of specific S4 complexes.Figure 3.


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

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

Dual-label competitive binding assay. (a) Scheme of the competitive binding experiments with two labeled RNAs. Complexes of the 5′ domain were separated on 8% polyacrylamide gels in TBE, while complexes of the 5WJ variants were separated on 6% polyacrylamide gels in TKM2, as described in ‘Material and Methods’ sections. (b) Distribution of S4 between 5′ domain and 5WJ RNAs. The counts in each complex and in free RNA were quantified and used to calculate Krel [Equation (2)]. For the 5WJ RNA, average Krel = 0.6 ± 0.1. The amount of labeled 5WJ complex decreases as unlabeled 5WJ RNA is added because its specific activity decreases. See Table 1 for further data.
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Related In: Results  -  Collection

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Figure 3: Dual-label competitive binding assay. (a) Scheme of the competitive binding experiments with two labeled RNAs. Complexes of the 5′ domain were separated on 8% polyacrylamide gels in TBE, while complexes of the 5WJ variants were separated on 6% polyacrylamide gels in TKM2, as described in ‘Material and Methods’ sections. (b) Distribution of S4 between 5′ domain and 5WJ RNAs. The counts in each complex and in free RNA were quantified and used to calculate Krel [Equation (2)]. For the 5WJ RNA, average Krel = 0.6 ± 0.1. The amount of labeled 5WJ complex decreases as unlabeled 5WJ RNA is added because its specific activity decreases. See Table 1 for further data.
Mentions: To evaluate whether our results were strongly biased by nonspecific binding of S4 (31), we also performed competitive binding experiments where both RNAs were 32P-labeled (Figure 3a). The advantage of this method is that the protein concentration is lower than the total RNA concentration, minimizing contributions from nonspecific interactions (34). To detect S4 binding to two labeled RNA substrates, an aliquot of each binding reaction was loaded on an 8% TBE gel to resolve the 5′ domain complexes, while another aliquot was loaded on a 6% TKM2 gel to resolve the smaller RNA complexes (Figure 3b). The results of the dual-label competition experiments were in good agreement with the competition assays done with only labeled 5′ domain and excess S4 (Table 1). Thus, we concluded that both sets of competition experiments were likely reporting the relative stabilities of specific S4 complexes.Figure 3.

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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