The phage HK022 Nun protein excludes phage by binding nascent and than at and may be the presence of RNase III processing sites (rIII) located immediately promoter distal to and rIII efficiently titrated Nun, allowing to grow on a strain that expressed Nun, whereas a transcript carrying only or templates. does not reflect the inability of N to compete with Nun at in vivo. A third conserved motif, box C (8 nt) (Fig. ?(Fig.1),1), lies downstream of and and does not appear to play a role in antitermination (5). The two sites differ in the spacer regions between box A, box B, and box C and by a single nt switch in the box B loop and the sixth nt in box C. Open in a separate window FIG. 1. Structures of and in vivo is usually both more efficient and less sensitive to mutations than termination 1243244-14-5 at (12, 18). mutations have been isolated that block Nun termination only at for that of did not increase the sensitivity of the changed transcript to the mutant (1). Washburn et al. (26) proposed that the phenotypic difference between and might 1243244-14-5 be explained by the relative distances of the two elements from their respective promoters. is 34 nt from lies 227 nt from its cognate promoter. True termination of Nun-arrested TEC requires host Mfd protein, a DNA helicase that recognizes and dissociates stalled RNAP. Mfd appears to take action at but not at precludes Mfd access. Thus, the off-rate of Nun-TEC complexes is lower at than at (26). also differs from in that it lies immediately promoter proximal to RNase III cleavage sites (rIII) (Fig. ?(Fig.1).1). Nascent (4, 10). Isolation of rIII mutants resistant to Nun inhibition of N translation (and gene from pYW1 (26) into the EcoRI and EcoRV sites of pZero-2 (Invitrogen, Carlsbad, CA) and the gene from pYW1 into the XhoI and XbaI sites. Plasmids pRSW110, pRSW111, pRSW112, pRSW113, and pRSW114 were produced by MADH3 cloning the [transcriptional fusion28????N9479N99 [transcriptional fusion28????N9480N9478(HK022)This study????N9481N9479(HK022)This study????N9482N9478/pBad-NunK107AThis study????N9483N9479/pBad-NunK107AThis study????N9484N9480/pACS21This study????N9485N9480/pSDF701This study????N9486N9481/pACS21This study????N9487N9481/pSDF701This study????N9488N9478/pACS21This study????RSW149N7723/pBad-NunK107AThis study????RSW237RSW149/pRSW110This study????RSW238RSW149/pRSW111This study????RSW258N9478/pSDF701This study????RSW279N7726/pACS21This study????RSW280N7726/pSDF701This study????RSW353RSW149/pRSW112This study????RSW354RSW149/pRSW113This study????RSW355RSW149/pRSW114This studyPlasmids????pBad-NunK107ApACYC1ori AmprpBad-NunK107A26????pACS21ColE1ori AmprRNAP and RNase III were purchased from Epicenter (Madison, WI). The Nun protein was a generous gift of Hyeong Kim. -Galactosidase assays. Strains were grown in LB at 32C with shaking to an optical density at 600 nm of 0.1 and then shifted to 42C and grown to an optical density at 600 nm of 0.6. Where indicated, the appropriate antibiotic (100 g/ml ampicillin or 50 g/ml kanamycin) was present. Nun, when expressed from a plasmid under the control of the pBAD promoter, was induced by the addition of 0.05% arabinose. Cells were assayed for -galactosidase activity as explained by Miller (14). Templates for in vitro transcription. DNA templates were generated by PCR, using AmpliTaq DNA polymerase (Roche Diagnostics, Branchburg, NJ) and DNA oligonucleotides (5-GGAATTCCATATGTCAGATCTCTCACCTACCAAAC-3 and 5-AGGGCGGTTAACTGGTTTTG-3) to amplify a 500-bp fragment of phage including (5-CCGTGATCAGCAGAAGGCTTTGCCCACACACATACGAAACGAAGC) were amplified using the indicated DNA oligonucleotides 1243244-14-5 paired with the oligonucleotide 5-GGAATTCCATATGTCAGATCTCTCACCTACCAAAC-3, digested with BclI, and ligated to the 3 fragment of produced with the oligonucleotides 5-AGGGCGGTTAACTGGTTTTG-3 and 5-CGGGATCCTTTGAATGCTGCCC-3 and digested with BamHI. produced with the oligonucleotides 5-AGGGCGGTTAACTGGTTTTG-3 and 5-CGGGATCCGCAGCTAATCCGGAATC-3 and digested with BamHI. produced with the oligonucleotides 5-AGGGCGGTTAACTGGTTTTG-3 and 5-CGGGATCCCACACACCCCAAAGC-3 and digested with BamHI. produced with the oligonucleotides 5-AGGGCGGTTAACTGGTTTTG-3 and 5-CGGGATCCGCAGCTAATCCGGAATTGCATTTACTGCTAATGCTTCG -3 and digested with BamHI. In vitro termination assay. Open up complexes were produced by preincubating 0.1 pmol template bound with 0.5 units RNAP (Epicenter) in 50 l TB (20 mM Tris-acetate [pH 7.9], 60 mM potassium acetate, 4 mM 1243244-14-5 magnesium acetate, 1 mm dithiothreitol, 0.25 mg/ml bovine serum albumin, and 5% glycerol) for 5 min at 32C. The Nun proteins, when included, was added at the indicated focus (1.25, 2.5, or 5 pmol/response). Transcription was initiated with the addition of a 10 M focus of every nucleoside triphosphate plus 1 Ci [-32P]ATP. After incubation at 32C for 5 min, the reactions had been terminated with the addition of 50 l end alternative (375 mM sodium acetate [pH 5.2] and 62.5 mM EDTA). The response mixtures had been extracted with the same level of phenol-chloroform-isoamylalcohol (Sigma) and ethanol precipitated with 3 volumes of 95% ethanol. Extracted RNAs had been after that resolved in a denaturing 12% polyacrylamide gel and analyzed by autoradiography. Readthrough transcription was measured by excising the correct gel bands and calculating radioactivity in a liquid.