Title |
Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction |
Author |
Matt N. Hicks1, Sanjiva Gunasekara1, Jose Serate2, Jin Park3, Pegah Mosharaf1, Yue Zhou1, Jin-Won Lee4, and Hwan Youn1* |
Address |
1Department of Biology, California State University Fresno, California 93740, USA, 2Department of Bacteriology, University of Wisconsin-Madison, Wisconsin 53706, USA, 3Department of Computer Science, California State University Fresno, California 93740, USA, 4Department of Life Science and Institute for Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea |
Bibliography |
Journal of Microbiology, 55(10),816-822, 2017,
|
DOI |
10.1007/s12275-017-7266-x
|
Key Words |
CRP, Escherichia coli, Gly184, DNA binding,
transcriptional activation, conformational flexibility |
Abstract |
The Escherichia coli cAMP receptor protein (CRP) utilizes the
helix-turn-helix motif for DNA binding. The CRP’s recognition
helix, termed F-helix, includes a stretch of six amino
acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185)
for direct DNA contacts. Arg180, Glu181 and Arg185 are
known as important residues for DNA binding and specificity,
but little has been studied for the other residues. Here
we show that Gly184 is another F-helix residue critical for
the transcriptional activation function of CRP. First, glycine
was repeatedly selected at CRP position 184 for its unique
ability to provide wild type-level transcriptional activation
activity. To dissect the glycine requirement, wild type CRP
and mutants G184A, G184F, G184S, and G184Y were purified
and their in vitro DNA-binding activity was measured.
G184A and G184F displayed reduced DNA binding, which
may explain their low transcriptional activation activity. However,
G184S and G184Y displayed apparently normal DNA
affinity. Therefore, an additional factor is needed to account
for the diminished transcriptional activation function in
G184S and G184Y, and the best explanation is perturbations
in their interaction with RNA polymerase. The fact that glycine
is the smallest amino acid could not fully warrant its suitability,
as shown in this study. We hypothesize that Gly184
fulfills the dual functions of DNA binding and RNA polymerase
interaction by conferring conformational flexibility
to the F-helix. |