Role of tyrosine residue 264 of RecA for the binding of cofactor and DNA.
The tyrosine fluorescence of the RecA protein is quenched by about 15% upon binding of the cofactor analog adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). This quenching is not observed with a modified RecA in which the tyrosine residue at position 264 (Tyr-264) is replaced for alanine by site-directed mutagenesis, a modification which also results in a decrease of binding affinity of cofactor. This indicates that Tyr-264 is responsible for the fluorescence change and that the residue is close to or within the cofactor binding site. Upon DNA binding, a change of tyrosine fluorescence is observed both with the modified protein and with wild type RecA, indicating that DNA binding affects the environment of other tyrosine residues than Tyr-264. However, the change is significantly smaller in the modified protein, suggesting that both Tyr-264 as well as other residue(s) may be affected by the DNA binding. Changed fluorescence properties of the remaining tyrosine residues as a result of a slightly different DNA binding mode of the modified protein are also possible. Tyr-264 may be an important residue for the allosteric effect induced by the cofactor for the binding of DNA to RecA. In the recent crystal structure of RecA-ADP published by Story and Steitz (Story, R.M., and Steitz, T. A. (1992) Nature 355, 374-376), ADP is stacked with Tyr-103 and does not interact with Tyr-264. The fact that we observe no interaction of ATP gamma S with Tyr-103 (as evidenced from absence of fluorescence change) but instead with Tyr-264 may suggest an important conformational difference between the RecA complexes with, respectively, ADP and ATP.
Eriksson
S
S
Department of Physical Chemistry, Chalmers University of Technology, Göteborg, Sweden.
Nordén
B
B
Morimatsu
K
K
Horii
T
T
Takahashi
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D005453:Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
C022571:adenosine 5'-O-(3-thiotriphosphate); D014443:Tyrosine; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases