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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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Competitive binding of Bst S4 to the 16S 5′ domain RNA. (a) S4 binding measured by gel mobility shift. 0.5 nM 32P-labeled and 0–400 nM unlabeled 5′domain RNAs were incubated with 31.5 nM Bst S4 in HKM4 buffer at 42°C. Gel is 8% polyacrylamide in TBE. NP, 32P-5′domain RNA only. (b) The fraction of complexed 32P-labeled 5′domain RNA versus competitor RNA was fit to Equation (1). Filled circles, 5′ domain (Kd, 5′domain = 5.5 ± 2.6 nM); filled squares, 16S rRNA (Krel = 1.6 ± 0.8).
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Figure 2: Competitive binding of Bst S4 to the 16S 5′ domain RNA. (a) S4 binding measured by gel mobility shift. 0.5 nM 32P-labeled and 0–400 nM unlabeled 5′domain RNAs were incubated with 31.5 nM Bst S4 in HKM4 buffer at 42°C. Gel is 8% polyacrylamide in TBE. NP, 32P-5′domain RNA only. (b) The fraction of complexed 32P-labeled 5′domain RNA versus competitor RNA was fit to Equation (1). Filled circles, 5′ domain (Kd, 5′domain = 5.5 ± 2.6 nM); filled squares, 16S rRNA (Krel = 1.6 ± 0.8).

Mentions: 32P-labeled E. coli 5′ domain RNA (nts 21–562 of 16S rRNA) was incubated with 0–400 nM Bst S4 at 42°C, and the S4–rRNA complex was detected by a native gel mobility shift (see ‘Materials and methods’ section). The apparent binding constant from direct titrations was 20 ± 4 nM, similar to the value obtained from nitrocellulose filter binding assays (Kd = 12 ± 5 nM; data not shown). When unlabeled 5′ domain RNA was added as a competitor, the Kd for the 5′ domain was determined to be 5.5 ± 2.6 nM (Figure 2). This is higher than the previously reported value of 0.7 nM for the specific complex, but much lower than the Kd for nonspecific binding (31). Kinetic dissociation experiments show that the electrophoretically retarded RNA likely contains a mixture of high- and low-affinity complexes that both contribute to the measured Kd (Bellur,D.L. and Woodson,S.A., in preparation). This may account for the discrepancy in Kd values and the small deviation of the binding data in Figure 2b from a two-state model. We consider the relative binding affinities obtained by competition more reliable than the absolute Kd values obtained by direct titration.Figure 2.


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

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

Competitive binding of Bst S4 to the 16S 5′ domain RNA. (a) S4 binding measured by gel mobility shift. 0.5 nM 32P-labeled and 0–400 nM unlabeled 5′domain RNAs were incubated with 31.5 nM Bst S4 in HKM4 buffer at 42°C. Gel is 8% polyacrylamide in TBE. NP, 32P-5′domain RNA only. (b) The fraction of complexed 32P-labeled 5′domain RNA versus competitor RNA was fit to Equation (1). Filled circles, 5′ domain (Kd, 5′domain = 5.5 ± 2.6 nM); filled squares, 16S rRNA (Krel = 1.6 ± 0.8).
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Figure 2: Competitive binding of Bst S4 to the 16S 5′ domain RNA. (a) S4 binding measured by gel mobility shift. 0.5 nM 32P-labeled and 0–400 nM unlabeled 5′domain RNAs were incubated with 31.5 nM Bst S4 in HKM4 buffer at 42°C. Gel is 8% polyacrylamide in TBE. NP, 32P-5′domain RNA only. (b) The fraction of complexed 32P-labeled 5′domain RNA versus competitor RNA was fit to Equation (1). Filled circles, 5′ domain (Kd, 5′domain = 5.5 ± 2.6 nM); filled squares, 16S rRNA (Krel = 1.6 ± 0.8).
Mentions: 32P-labeled E. coli 5′ domain RNA (nts 21–562 of 16S rRNA) was incubated with 0–400 nM Bst S4 at 42°C, and the S4–rRNA complex was detected by a native gel mobility shift (see ‘Materials and methods’ section). The apparent binding constant from direct titrations was 20 ± 4 nM, similar to the value obtained from nitrocellulose filter binding assays (Kd = 12 ± 5 nM; data not shown). When unlabeled 5′ domain RNA was added as a competitor, the Kd for the 5′ domain was determined to be 5.5 ± 2.6 nM (Figure 2). This is higher than the previously reported value of 0.7 nM for the specific complex, but much lower than the Kd for nonspecific binding (31). Kinetic dissociation experiments show that the electrophoretically retarded RNA likely contains a mixture of high- and low-affinity complexes that both contribute to the measured Kd (Bellur,D.L. and Woodson,S.A., in preparation). This may account for the discrepancy in Kd values and the small deviation of the binding data in Figure 2b from a two-state model. We consider the relative binding affinities obtained by competition more reliable than the absolute Kd values obtained by direct titration.Figure 2.

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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