−10 and −35 sequences in <i>RNAIp</i>, <i>RNAIIp</i>, and DR.

<p>(A) Sequences of -10 and -35 boxes in <i>RNAIp</i> and <i>RNAIIp</i>. (B) Forty-one copies of 15-bp repeats in DR region, from nt 3341 to nt 3955 in pSW200. Sequences that are homologous to the -35 box are underlined.</p

Binding of RNA polymerase to 15-bp repeats.

<p>(A) Sequences of DNA probes used in EMSA. DR probe contains four copies of 15-bp repeats from nt 3896 to nt 3955. Sequences highlighted in DR probe resemble <b>−</b>35 sequences. Underlined region in DR probe from nt 3900 to nt 3930 was inserted into 167 sequence to yield 167-DR. (B) Purified RNA polymerase holoenzyme (Epicentre) was added to a reaction mixture that contained biotinylated DR probe (lanes 1–7). Unlabeled DR probe was added to compete for binding (lanes 5–7). Probe 167 and 167-DR (lane 8–11) were used to confirm binding of RNA polymerase to repeat region. Protein-DNA complex was separated using a 7% polyacrylamide gel and detected using a LightShift chemiluminescence EMSA kit (Pierce).</p

Exclusion of pSW207 by pSW201 derivatives.

<p>Exclusion of pSW207 by pSW201 derivatives.</p

Competition of ColE1-like plasmids by pSW200 repeats.

<p>(A) Three plasmids in the ColE1 family that contains DR – pBR322-210, pACYC-210, and pSW100-210 were tested to determine their capacity to destabilize an incompatible plasmid. <i>E. coli</i> HB101 was transformed with pUC18 (lane 1), pBR322-210 (lane 2), pACYC184 (lane 4), pACYC-210 (lane 5), pSW106 (lane 7), and pSW100-210 (lane 8). The cells were also cotransformed with pUC18 and pBR322-210 (lane 3), pACYC184 and pACYC-210 (lane 6), and pSW106 and pSW100-210 (lane 9). (B) Plasmids pRK-210, a plasmid that contains an RK2 replicon and DR, and pACYC-210 were tested to determine their ability to destabilize a compatible plasmid. <i>E. coli</i> HB101 was cotransformed with pRK-210 and pSW207 (lane 1), pACYC-210 and pSW207 (lane 2), and pRK-210 and pACYC184 (lane 3). Plasmids were isolated using an alkaline lysis method and detected by agarose gel electrophoresis. Asterisks indicate pUC18, pACYC184, and pSW106.</p

Correlation between number of repeats and competition among plasmids.

<p>Derivatives of pSW201 that contain various numbers of 15-bp repeats were cotransformed with pSW207 into <i>E. coli</i> HB101. The transformants were plated on LB agar and then replica-plated on LB agar that contained Km or Tc to select those that contained pSW201 derivatives with various numbers of repeats (empty column) and pSW207 (filled column).</p

Exchange of RNA polymerase from DR region to <i>RNAIp</i> and <i>RNAIIp</i>.

<p>(A) Sequences of <i>RNAIp</i> and <i>RNAIIp</i> probes. (B) Purified RNA polymerase (RP) was added to ³²P-labeled-DR probe (Fig. 7A). Unlabeled <i>RNAIIp</i> and <i>RNAIp</i> probes, which contained sequences shown in (A), were used to analyze exchange of RNA polymerase from DR region to <i>RNAIp</i> and <i>RNAIIp</i>. (C) DNA-protein complexes that contained a biotinylated-DR probe (Bio-DR) and RNA polymerase (Bio-DR/RP) were captured using streptavidin-coated magnetic beads. After unbound RNA polymerase had been removed, ³²P-labeled <i>RNAIIp</i> and <i>RNAIp</i> probes were added to RNA polymerase-DR complex to analyze exchange of RNA polymerase from repeats to <i>RNAIp</i> and <i>RNAIIp</i>. Probe 167 was used as a negative control.</p

Transcription from DR and <i>RNAIIp</i>.

<p>Transcriptional fusion was generated by inserting a fragment from nt 1 to 426, which contains <i>RNAIIp</i> in pSW201 (pSW242), and a fragment from nt 3356 to 4367 in pSW201, which contains DR (pSW243), into a luciferase reporter plasmid, pKK175-6-lux. Luciferase activity was monitored with a luminometer and presented in relative light units (RLU). Each experiment was performed three times and each sample in the experiment was prepared in duplicate.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

RT-qPCR analysis of activity of <i>RNAIp</i> and <i>RNAIIp</i>.

<p>(A) Map of pSW261, pSW262, pSW263, and pSW264. Arrow indicates direction of transcription. Numbers represent relative nucleotide positions in pSW200. (B)(C) RNA that was transcribed from a region in Tc-resistance gene was amplified by RT-qPCR. 16S rRNA was used as an internal control and amounts of mRNA that were transcribed from <i>RNAIp</i> and <i>RNAIIp</i> were normalized to amount of 16S rRNA. Amount of <i>tet</i> mRNA from pSW261 and pSW263 was set to 100%. Experiment was performed three times and each sample was prepared in duplicate. Empty square: a fragment from tetracycline-resistance gene; error bar: standard deviation.</p

Involvement of MCAF1 in the interaction between ATF2 and Rta.

<p>(A) His-tagged proteins, including Rta, ATF2, and MCAF1, were expressed in E. coli BL21(DE3). Lysates were mixed in different combinations for immunoprecipitation (IP) using anti-ATF2 antibody, and proteins bound to protein A/G agarose beads were then detected by immunoblotting (IB) using anti-Rta or anti-ATF2 antibodies. Lane 1 was loaded with 1% His-Rta. The reaction was also conducted using anti-IgG as a control. (B) 293T cells were cotransfected with pCMV-R and either MCAF1 siRNA or control siRNA. After transfection, cells were immunoprecipitated (IP) by anti-ATF2 (lanes 4, 6) or anti-IgG antibody (lanes 3, 5), and detected by immunoblotting (IB) using anti-Rta antibody. The effect of MCAF1 siRNA on the expression of MCAF1 was examined by immunoblotting using anti-MCAF1 and anti-ATF2 antibodies (lanes 1, 2). Lanes 1 and 2 were loaded with 1% of total protein from cell lysates.</p