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KMID : 0578320080260010026
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Molecules and Cells
2008 Volume.26 No. 1 p.26 ~ p.33
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The Terminal and Internal Hairpin Loops of the ctRNA of Plasmid pJB01 Play Critical Roles in Regulating Copy Number
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Kim Sam-Woong
Eo Seong-Kug
Kang Ho-Young
Lee John-Hwa
Jeong In-Sil
Jeong Eun-Ju
Tak Je-Il
Bahk Jeong-Dong
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Abstract
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The plasmid pJB01, a member of the pMV158 family isolated from Enterococcus faecium JC1, contains three open reading frames, copA, repB, and repC. Plasmids included in this family produce counter-transcribed RNA (ctRNA) that contributes to copy number control. The pJB01 ctRNA, a transcript which consists of 54 nucleotides (nts), is encoded on the opposite strand from the copA/repB intergenic region and partially overlaps an atypical ribosome binding site (ARBS) for repB. The ARBS is integrated by the two underlined conserved regions: 5’-TTTTTGTNNNNTAANNNNN NNNNATG-3’, and the ctRNA is complementary only to the 5’ conserved sequence 5’-TTTTTGT-3’. This complementary sequence is located at a distance from the terminal loop of the ctRNA secondary structure. The ctRNA structure predicted by the mfold program suggests the possible generation of a terminal and an internal hairpin loop. The amount of in vitro translation product of repB mRNA was inversely proportional to the ctRNA concentration. Mutations in the terminal and internal hairpin loops of the ctRNA had inhibitory effects on its binding to the target mRNA. We propose that the intact structures of the terminal and internal hairpin loops, respectively, play important roles in forming the initial kissing and extending complexes between the ctRNA and target mRNA and that these regulate the copy number of this plasmid.
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KEYWORD
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ARBS, ctRNA, internal and terminal loops, operon, pJB01, RepB
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