Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.
Psu, a coat protein from bacteriophage P4, has been shown to inhibit Rho-dependent transcription termination in vivo. Co-overexpression of Psu and Rho led to the loss of viability of the cells, which is the consequence of the anti-Rho activity of the protein. The antitermination property of Psu is abolished either by the deletion of 10 or 20 amino acids from its C terminus or by a mutation, Y80C, in Rho. All these experiments indicated probable interactions between Rho and Psu. Purified Psu protein is alpha-helical in nature and appeared to be a dimer. Co-purification of Rho and wild-type Psu on an affinity matrix and co-elution of both of them in Superose-6 gel filtration suggests a direct association of these proteins, whereas a C terminus 10-amino acid deletion derivative of Psu failed to be pulled down in this assay. This indicates that the loss of the function of these mutants is correlated with their inability to interact with each other. In vitro termination assays revealed that Psu can inhibit Rho-dependent termination specifically in a concentration-dependent manner. The presence of Psu affected the affinity of ATP and reduced the rate of ATPase activity of Rho but did not affect either primary or secondary RNA binding activities. In the presence of Psu, Rho was also observed to release RNA very slowly from a stalled elongation complex. We propose that Psu inhibits Rho-dependent termination by slowing down the translocation of Rho along the RNA because of its slow ATPase activity.
Pani
Bibhusita
B
Laboratory of Transcription Biology, Centre For DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad-500076, India.
Banerjee
Sharmistha
S
Chalissery
Jisha
J
Muralimohan
Abishek
A
Abishek
Muralimohan
M
Loganathan
Ramya Malarini
RM
Suganthan
Ragan Babu
RB
Sen
Ranjan
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D003090:Coliphages / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D046912:Multiprotein Complexes; D009154:Mutation; D011485:Protein Binding; D012234:Rho Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D036022:Capsid Proteins; D046912:Multiprotein Complexes; D012234:Rho Factor; C074513:polarity suppression factor, bacteriophage P4; D000255:Adenosine Triphosphate