Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.
The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.
Tabuchi
A
A
Department of Bacteriology, Shinshu University School of Medicine, Matsumoto, Japan.
Ohnishi
M
M
Hayashi
T
T
Terawaki
Y
Y
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D010957:Plasmids / Q000145:classification / Q000235:genetics*; D011485:Protein Binding; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin; D013329:Structure-Activity Relationship; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases