We have isolated two mutant plasmid derivatives of ColEl that exhibit temperature-sensitive replication properties. Both mutants have a normal copy number at 30°C but increase their copy number 30-to 40-fold after a shift in temperature to 42TC. A plasmid-encoded enzyme, &3-actamase (penicillinase, EC 3.5.2.6), undergoes a 30-to 40-fold increase in specific activity concomitant with the increase in plasmid copy number. The cop' phenotype of these mutants is not due to the synthesis of a temperature-sensitive polypeptide. Both mutations are located in an untranslated region of the plasmid genome encoding two overlapping transcripts involved in plasmid replication: a small transcript known as RNA I that acts as a negative control element in replication and a large transcript that has been characterized as the replication primer in vitro. The mutations alter the sequence encoding the primer but lie immediately 5' to the initiating nucleotide of RNA I, in the RNA I promoter region. The possibility that the temperature-dependent plasmid DNA amplification is a consequence of a temperature-sensitive RNA I promoter was tested by inserting the RNA I promoters from the wild-type and mutant plasmids into a plasmid in which galactokinase expression is dependent upon an exogenous promoter. These experiments demonstrate that the mutant promoters are not temperature-sensitive. Rather, the mutations may affect the secondary structure of the replication primer in a region important for RNA I interaction.Extrachromosomal elements such as the plasmid ColEl are model systems for the study of DNA replication control mechanisms because of their small size, genetic dispensibility, and the fact that they encode information which plays a role in the maintenance of a characteristic and stable copy number (1). Mutations in ColEl derivatives that display elevated copy numbers (designated cop-) have been isolated (2, 3). These mutants increase the copy number from about 15 copies per chromosome to 200-300 per chromosome (2, 3). The behavior of the recessive copj plasmid pOP1A6 was consistent with the existence ofa diffusible, replicon-specific inhibitor ofplasmid DNA replication. We have shown that the mutation in pOP1A6 is a single base pair (bp) change in a region encoding a small, nontranslated RNA molecule designated RNA I (4). RNA I is 108 nucleotides long and is transcribed from a region 450 bp from the origin of replication (5).The simple view that RNA I was a trans-acting, replicon-specific repressor was complicated by the discovery that the region of the genome encoding RNA I also encodes a RNA primer of DNA replication in vitro (6). Thus, the mutation described above alters the sequence of both RNA I and the in vitro replication primer. This primer is 555 nucleotides long and is transcribed from the DNA strand complementary to that encoding RNA I. Synthesis ofthe primer transcript initiates near the termination site of RNA I. Using an in vitro DNA replication system, Itoh and Tomizawa (6) have shown that generation of pri...
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