Here we report on the selection and characterization of RNA aptamers that recognize E. coli ribosomal protein S7. Ribosomal protein S7 plays two important roles in ribosome biogenesis: (1) as an assembly initiator, S7 nucleates the folding of the 3' major domain of 16S rRNA, and (2) it binds to the str operon and represses the translation of S12, S7, and EF-G. The primary and secondary structures of the S7 binding sites of rRNA and mRNA share limited sequence and structural homology and the required elements for high affinity binding have not been entirely elucidated. We have selected RNA aptamers that share very little primary sequence homology to either the S7 binding site of 16S rRNA or to the intercistronic region of str mRNA. Many of the aptamers are expected to fold into three-helix junctions, a structure particularly reminiscent of the mRNA. Interestingly, the aptamers exhibit cooperative binding with Hill coefficients of ∼3 indicating that they are detecting a quaternary structure of S7.
We have found that the S7 aptamers use the same amino acids and structural elements to bind S7 as the rRNA and mRNA indicating that the same binding site is used for all three RNAs. With gel filtration, we were only able to isolate the aptamer/S7 complex at a 1:1 stoichiometry, indicating that the proposed quaternary structure of S7 is weak. However, deletion of the β-ribbon nearly eliminates cooperative aptamer binding suggesting that this structural element may be involved in protein-protein interaction. Furthermore, pre-treatment of native S7 with the N-terminal extension also results in a significant reduction in cooperative aptamer binding.
The results presented here suggest that S7 itself may undergo conformational rearrangement subsequent to 16S rRNA binding, and may help explain the strong temperature-dependent rearrangements at the binding site of S7 within the 16S rRNA. Furthermore, we propose that the weak, multimeric interaction of S7 may have a role in the retroregulation of S12. S7 may bind to the mRNA in a pre-multimerized form or multimerize subsequent to binding, resulting in ribosome stalling due to the multimeric obstacle. If the S7/S7 interaction is weak however, then it may be easily disrupted by repeated ribosome bombardment, causing eventual decay of the multimer and relieving some of the translational repression. Translational repression of the genes encoding S7 and EF-G would remain constant over time however, because the monomeric S7 bound more tightly to the intercistronic region would continue to prevent translational coupling with the upstream gene encoding S12.
|Commitee:||Culver, Gloria M., Miller, W. Allen, Minion, F. Chris, Wannemuehler, Michael J.|
|School:||Iowa State University|
|Department:||Biochemistry, Biophysics, and Molecular Biology|
|School Location:||United States -- Iowa|
|Source:||DAI-B 70/08, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology, Biochemistry|
|Keywords:||Aptamers, Cooperative binding, Isothermal titration calorimetry, Quaternary structure, Ribosomal protein, S7, Translational regulation|
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