Unusual mRNA pseudoknot structure is recognized by a protein translational repressor

Area of Science:

• Molecular Biology
• RNA Structure
• Bacterial Gene Regulation

Background:

• Ribosomal protein S4 regulates translation of ribosomal proteins in the E. coli alpha operon.
• S4 specifically binds an RNA fragment at the translational initiation site.
• Previous studies suggested a pseudoknot structure for the S4 binding site.

Purpose of the Study:

• To systematically test and confirm the proposed pseudoknot structure of the S4 binding site.
• To investigate the role of RNA structure in translational repression by S4.

Main Methods:

• Site-directed mutagenesis to create compensatory base pair changes.
• S4 binding assays to measure the effects of mutations.
• RNA structure mapping experiments.

Main Results:

• The proposed pseudoknot structure of the S4 binding site was confirmed.
• Two additional, unexpected interactions within the pseudoknot were identified.
• An unusual "double pseudoknot" structure was characterized, linking upstream hairpin with downstream sequences.

Conclusions:

• The double pseudoknot RNA structure is essential for S4 binding.
• Stabilization of this double pseudoknot by S4 accounts for translational repression of the alpha operon.
• This study provides a detailed structural basis for translational control in bacteria.