Current Protocols in Molecular Biology - Chapter 11

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Cohn and K. ) pp. 169-194. Academic Press, San Diego. F. W. 1983. Viability of λ phages carrying a perfect palindrome in the absence of recombination nucleases. Nature 305:448. , and Monod, J. 1967. Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the β-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24:339-343. V. 1995. Structural and mechanistic studies of galactoside acetyltransferase, the Escherichia coli LacA gene product.

The copy number of ColE1 plasmids is determined by the balance between successful RNAII processing events and those inhibited by RNAI. Adapted from Gerhart et al. (1994) with permission from the Annual Review of Microbiology. 3 Current Protocols in Molecular Biology Supplement 41 the RNAI/RNAII interaction result in a higher plasmid copy number. For example, the pUC plasmid series have pMB1 replicators that do not contain an intact rop gene, which accounts for a 2-fold increase in copy number over plasmids with intact pMB1 replicons.

Both ColE1 and pMB1 plasmids are highcopy-number plasmids, maintained at between 15 and 25 copies per bacterial cell, respectively. The copy number of these plasmids is regulated by an antisense RNA transcript, RNAI, and the protein ROP, the product of the rop gene. RNAI and the ROP protein act in concert to intercept formation of the proper RNAII secondary structure. RNAI is exactly complementary to the 5′ end of the RNAII transcript. RNAI base pairs with the 5′ end of RNAII, preventing the formation of the specific secondary structure necessary for establishment of a persistent hybrid between RNAII and the DNA template that is a prerequisite for maturation of the RNAII primer.

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