Regulation of plasmid pE194 replication: control of cop-repF operon transcription by Cop and of repF translation by countertranscript RNA

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Abstract
The cop-rep region of plasmid pE194 contains two tandem structural genes, cop and repF, as well as the plus and minus origins of replication. The two structural genes comprise an operon whose expression is repressed by the binding of Cop protein to a 28-bp inverted complementary repeat sequence that overlaps the cop-repF promoter. From its position relative to the promoter and the experimentally determined footprint made by the Cop protein, the 28-bp inverted complementary repeat sequence is presumed to function as the cop operator. The intercistronic region between cop and repF is 80 nucleotides (nt) long and is transcribed bidirectionally: in the forward direction as part of the synthesis of the cop-repF message (ca. 900 nt), and in the reverse direction to yield a countertranscript ca. 65 nt long. The proposed countertranscript RNA (ctRNA) can form a single stem-and-loop structure that includes the single SphI sequence of plasmid pE194 as part of the loop-forming segment. Enlargement of the proposed loop from 6 to 14 nt by insertion of a SphI-BamHI adapter at the SphI site or contraction of the proposed loop down to 4 nt, by cutting with SphI followed by blunting with S1 nuclease, yields mutants with an increased copy number. By gel retardation and DNaseI footprinting analysis, Cop protein was shown to bind to the promoter region of cop; no binding by Cop protein at the 5' end of repF was detected. Two major transcripts were synthesized in vitro by using cop-repF region DNA as a template, the tandem cop-repF transcript, and the ctRNA. Addition of purified Cop protein to an vitro transcription reaction mixture reduced only the rate of cop-repF transcription but not that of ctRNA. These observations suggest that regulations of repF occurs at two levels: (i) with Cop protein acting as a repressor of cop-repF mRNA transcription and (ii) with ctRNA acting as a repressor of RepF translation.