Abstract
Promoter escape is the final step of transcription initiation when RNA polymerase
(RNAP) has to relinquish its interactions with the promoter DNA before entering the elongation
phase. The efficiency of escape is dependent on two factors: one, the rate of escape, and two, the
extent of RNAP partitioning into the escape-competent productive open complexes. Both factors
are governed by the promoter sequence; thus, the efficiency of escape varies from promoter to
promoter. As examples, the T5 phage N25 promoter exhibits a high rate of promoter escape as
illustrated by a half-life of ~3 min for the production of full-length RNA whereas N25anti, a
promoter derived by mutating the initial transcribed sequence (ITS) of N25, exhibits an
extremely low rate of promoter escape with a half life ~45 min. However, N25anti yields
productive RNA at a more highly elevated level compared to N25; this was unexpected. Our
research team is currently investigating the cause of this unusually large variation in the amounts
of full- length RNA produced from these two promoters.
One explanation to account for the high level of productive RNA from the very slowescaping N25anti promoter could be that during transcription, some of the unproductive open
complexes (RPo’) reverted back to their closed conformation (RPc) (through the k-2 step) which
might then re-partition into the productive RPo that can subsequently undergo escape. If this is
true for N25anti, we might also see similar pattern of increasing full-length RNA production for
N25 in a long time course. However, our results with N25 promoter show that the plateau level
of full-length RNA stays constant over time, suggesting a negligible k-2 for this promoter. We
are currently undertaking the determination of k-2 for N25 and N25anti promoters by a different
method.
Another important factor in transcription is the KCl concentration. In a steady- state
transcription reaction, high salt concentration (i.e. 200 mM for the N25 promoters) has long been
correlated with stable open complex formation and higher productive yield. To examine if KCl
concentration plays a role in the behavior of N25 particularly in the production of full-length
RNA, single cycle time course transcriptions in various concentrations of KCl, were set up. We
found that at lower salt concentrations the rate of escape for N25 became faster and more
efficient. This result was directly opposite from what was expected from the steady-state
transcription results. Currently, we do not have a good explanation for this discrepancy, which
can only be resolved through further investigation.
Materials and Methods
Promoter sequence (template)
The promoter templates, N25 (-C) and N25 anti (-A) ,were provided by prof. Hsu. The ITS sequence of the
N25-anti promoter had been modified such that the first 40 sequence of its ITS lacked the nucleotide
adenosine (A).
Plasmid –cloning and purification using mini-prep
The plasmids with the promoter sequences were cloned in E. coli. The bacterial cells, pSAN1 and
pSAN5 were each added to LB broth (3.5mL) with ampicillin (35uL). The tubes were then incubated at
370C to let the cells grow overnight.
Qiagen mini-prep was used to isolate plasmid DNA. Cells were collected by centrifugation and
lysed in lysis buffer, and neutralized by adding neutralizing buffer to obtain a clear lysate. The plasmid
DNA was recovered from a Qiagen column from 50uL of TE buffer (10mM Tris-HCl, pH 8, 1mM
EDTA).
Agarose gel
Agarose gel was electrophoresis was performed to determine the size of the plasmid DNA : 1%
for 500-3000bp. The gel solution was prepared by adding 1g of Agarose in 100mL of 1X TAE buffer,
(40mM Trisma base, 20mM acetic acid, 1mM Na2EDTA, pH 7.4) with 0.5ug/uL of Ethidium bromide
and the same buffer was used for electrophoresis.
Polymerase Chain Reaction (PCR)
For 200ul PCR reaction, 2uL of 10ng/uL DNA, 20uL of 10X Thermo buffer, 12ul of 2.5mM of
MgCl2, 20uL of 2.5mM NTP, 20uL of 508 (u)Xba (2.5uM upstream primer; 5’ ATT TCT AGA CCT
TCC CGT TTG GC 3’), 20uL of 508 (d)Bam (2.5uM downstream primer; 5’ GGC GGA TCC CTA ACT
CCC CCC 3’) and 106uL of ddH2O were mixed together. To this mixture, 1uL of Taq DNA polymerase
was added. PCR amplification was carried out for 30-35 cycles where each cycle included incubation for
1 minute at 940C to denature the template strands, 1minute at 530C to anneal the primers and 1minute at
720C to extend the primers. Agarose gel electrophoresis (2%) was performed to check the amplification.
DNA recovery and clean up
In order to recover the PCR amplified DNA,
Single cycle transcription
Gel electrophoresis
Quantitation
Results
Discussions.