Masters Thesis

Transcriptional activation activity of camp receptor protein is sensitive to the size of its position-183 amino acid

The Escherichia coli cAMP receptor protein (CRP) requires both DNA binding and RNA polymerase recruitment for its transcriptional activation function. The DNA recognition of CRP is performed by the F-helix (residues 180-185) and several F-helix residues (Arg180, Glu181, Gly184, Arg185) are well-characterized as to their importance in DNA binding. However, no apparent function has been known for the other two residues (Thr182 and Val183). In this work, I show that the position-183 amino acid (Val 183) is important for the transcriptional activity of CRP. First, Ala substitution at position 183 led to an increase in transcriptional activation activity. Second, CRP mutants altered at position 183 were created to show an inverse correlation between amino acid size and transcriptional activation activity (V183A > V183G > wild type CRP > V183I > V183M > V183F = no activity). Third, the loss of the activity in V183F could be restored by a smaller amino acid substitution (IleGly) at position 172, implying that it is the combined size of positions 172 and 183 which is important. A structural analysis of CRP reveals that the position-183 amino acid faces the opposite of target DNA and is in close proximity to the position-172 amino acid. The requirement of a small residue at position 183 is hypothesized to avoid steric hindrance to the position-172 residue as steric hindrance would misalign the nearby AR1 residues, leading to poor RNA polymerase recruitment and transcriptional activation.

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