A novel indirect sequence readout component in the E. coli cyclic AMP receptor protein operator

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The cyclic AMP receptor protein (CRP) from Escherichia coli is a well-studied transcription factor.
• CRP dimer recognizes a consensus DNA sequence through direct amino acid-nucleobase interactions.
• Crystal structures reveal kinks at TG/CA steps near the 6-base-pair (N6) spacer, but its role is not fully understood.

Purpose of the Study:

• To systematically investigate the role of the N6 spacer sequence variability in CRP-DNA interaction.
• To determine how N6 spacer sequences influence CRP binding affinity to the lacP1 site.

Main Methods:

• In vitro selection system utilizing a randomized N6 spacer region.
• Analysis of CRP binding affinity to DNA with varied N6 sequences.
• Uranyl (UO2(2+)) probing to assess DNA structure and deformability.

Main Results:

• CRP binding to the lacP1 site can be enhanced up to 14-fold or completely abolished by altering N6 spacer sequences.
• Sequence analysis and uranyl probing data suggest a mechanism involving N6 spacer deformability.

Conclusions:

• The N6 spacer region is a critical determinant of CRP-DNA binding affinity.
• The deformability of the N6 spacer plays a significant role in the CRP-DNA interaction mechanism.