Primer extension analyses of RNA generated in vitro were performed according to manufacturer’s instructions (Promega) by using AMV Reverse Transcriptase. A sample (5 μl) of the in vitro transcription reaction was added to 6 μl of primer mixture containing 2 × AMV Primer Extension buffer and 2 pmol 32P-labeled primer, which annealed +103 bp downstream of the +1 transcriptional start site. Aliquots were electrophoresed on 8% (wt/vol) polyacrylamide, 7 M urea denaturing gels for 4000 V-h in × TBE. Densitometry and quantification were performed as described above.
In vivo RNA was obtained from WN310 containing pMLH17 and pCMW75 or pCMW98 grown in LB with 100 μg/ml ampicillin and 100 μg/ml kanamycin at 37°C with shaking at 220 rpm to an OD600 of ∼0.5. Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions. After elution, 5 μg total RNA in 5 μl was added to the primer mixture and subsequent steps for primer extension reactions were performed as described above.
In vivo RNA was obtained from WN310 containing pMLH17 and pCMW75 or pCMW98 grown in LB with 100 μg/ml ampicillin and 100 μg/ml kanamycin at 37°C with shaking at 220 rpm to an OD600 of ∼0.5. Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions. After elution, 5 μg total RNA in 5 μl was added to the primer mixture and subsequent steps for primer extension reactions were performed as described above.