Coordinated regulation of gene expression by E ... - Jacobs University
Coordinated regulation of gene expression by E ... - Jacobs University
Coordinated regulation of gene expression by E ... - Jacobs University
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MATERIALS AND METHODS<br />
free water (Ambion) for subsequent use in the primer extension reaction (10 μl for each<br />
primer).<br />
2.2.17 Primer extension<br />
The mRNA from in vitro transcription <strong>of</strong> ∆61 tyrT constructs and from tyrT with<br />
different supercoiling was divided into two 10 μl parts to be used for primer extension<br />
with Ex103 (for tyrT transcript) and bla primers. Primer extension from total RNA from<br />
LZ strains transformed with different tyrT plasmids was performed with Ex103 and a<br />
collective primer for chromosomal rrn operon’s P1 promoter, G11, as described <strong>by</strong><br />
Nasser et al.,[Nasser et al., 2001].<br />
The primers (200 pmol) to be extended were radioactively labeled using 50 μci<br />
<strong>of</strong> P32 γ-ATP (Amersham Biosciences now a part <strong>of</strong> GE Healthcare) and T4<br />
polynucleotide kinase (PNK) (Invitrogen) in a reaction volume <strong>of</strong> 20μl and the labeled<br />
primer purified using G25 spin columns (GE healthcare, formerly Amersham<br />
Biosciences). Primer extension reaction was performed from 3 μg <strong>of</strong> total RNA or<br />
mRNA from in vitro transcription both in a volume <strong>of</strong> 10 μl, 2 mM final concentration<br />
<strong>of</strong> dNTP mix, 2 pmol <strong>of</strong> labeled primer, 2 μl <strong>of</strong> 0.1M DTT, 4 μl <strong>of</strong> 5X reverse<br />
transcriptase buffer and 1 μl <strong>of</strong> LMV-reverse transcriptase. The primer extension<br />
products were separated on a 6% polyacrylamide gel and visualized <strong>by</strong><br />
phosphorimaging (FUJI film FLA-3000 phosphorimager).<br />
2.2.18 Potassium permanganate footprinting<br />
The components <strong>of</strong> the footprinting reaction are the same as the in vitro transcription<br />
reaction. The footprinting was carried out as described earlier [Nasser et al., 2001]. The<br />
addition <strong>of</strong> potassium permanganate (KMnO4), which oxidize preferentially the<br />
thymine nucleotide (cytosine can also be oxidized) <strong>of</strong> an open double helix, gives a<br />
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