The structure and function of the replication initiator protein (Rep) of pSC101: an analysis based on a novel positive-selection system for the replication-deficient mutants.
Plasmid pSC101 encodes a 37.5 kDa Rep (RepA) protein, which binds to three 21-base repeats (DR-1, DR-2, and DR-3) in the replication origin region (ori) of the plasmid to initiate replication. Rep also binds to two palindromic sequences (IR-1 and IR-2) which overlap the rep promoter. The binding of Rep to IR-2 represses the production of Rep itself. It is highly likely that the balance of these functions of Rep plays a major role in controlling the copy number of pSC101. In this study, we developed a positive-selection system for replication-deficient mutants of the initiator protein. This system can be applied to the study of other replication systems by changing ori and rep of pSC101 to the corresponding genes. Thirty-four replication-deficient (Ini(-)) mutants were isolated with this system, and analyzed as to the relation between the structure and function of the Rep protein. Seventeen of these 34 Ini(-) mutants were found to lack auto-repressor activity as well as initiator activity. DNA sequence analysis showed that one-third (from the C-terminus) of Rep is dispensable for the auto-repressor activity, while the initiator activity seems to require the whole protein.
Fueki
T
T
Institute for Gene Research, Kanazawa University, Kanazawa 920-0934, Japan. yamaguti@kenroku.kanazawa-u.ac.jp
Yamaguchi
K
K
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D020125:Mutation, Missense / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics* / Q000502:physiology; D018741:Replication Origin / Q000235:genetics*; D012641:Selection, Genetic; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases