How Does Rop Work?
Plasmids of the ColEl family provide a convenient model system for the investigation of the molecular mechanisms which regulate initiation of DNA replication in Escherichia coli. ColEl replicates in bacteria at a copy number of approximately 15. The observations that this copy number could be increased by mutagenesis and that some of these mutants are complemented in trans by plasmids of the same incompatibility group suggest that ColEl replication is negatively regulated (1–6). An attractive model of the processes of initiation of DNA replication was proposed by Tomizawa and coworkers who were able to reassemble from purified enzymes an in vitro system which can carry out the biochemical steps involved in primer formation and initial deoxyribonucleotide polymerization (3, 7). When origin sequences are incubated in the presence of RNA polymerase, two main transcripts can be identified (Figure 1). The longer one (RNA2) starts from a promoter 550 nucleotides upstream from the replication origin and is believed to form a hybrid with the DNA template in the origin region. This hybrid is processed by RNAseH to yield an RNA molecule of 550 nucleotides which can serve as primer for deoxyribonucleotide polymerization by DNA polymerase I.
KeywordsUrea Recombination Electrophoresis Polyacrylamide Tetracycline
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