Single-stranded RNA recognition by the bacteriophage T4 translational repressor, regA

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RegA is a T4 translational repressor impacting multiple T4 messages.
• RegA functions by blocking ribosome binding near mRNA AUG start codons.
• Affected mRNAs lack obvious similarities beyond the AUG start codon.

Purpose of the Study:

• To identify high-affinity RNA ligands for RegA using SELEX.
• To determine the specific RNA sequence and structural features recognized by RegA.
• To elucidate the mechanism of translational repression by RegA.

Main Methods:

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to isolate RNA ligands.
• RNA sequence analysis to identify conserved motifs.
• Chemical modification and nuclease digestion to probe RNA structure and binding sites.
• Boundary and footprinting experiments to define the RegA-binding site.

Main Results:

• SELEX yielded RNA ligands with a consensus sequence 5'-AAAAUUGUUAUGUAA-3'.
• The AUG start codon was invariant, indicating its primary role in binding specificity.
• Conserved elements include a UU motif 5' to the AUG and an upstream poly(A) tract.
• RegA binds to a single-stranded RNA region, suggesting discrimination based on primary sequence.
• Accumulated mutations in the consensus sequence significantly impact RegA binding.

Conclusions:

• RegA utilizes a specific primary RNA sequence, including the AUG start codon and flanking elements, for target recognition.
• RegA's binding site is single-stranded, indicating sequence-specific, rather than structure-specific, repression.
• RegA-mediated translational repression occurs sequentially, affecting messages until the regA gene itself is repressed.