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25 61 RNA Polymerase Structure

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25 61 RNA Polymerase Structure
wea25324_ch06_121-166.indd Page 122 11/13/10 6:14 PM user-f469
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6.1
/Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefiles
Chapter 6 / The Mechanism of Transcription in Bacteria
RNA Polymerase Structure
As early as 1960–1961, RNA polymerases were discovered
in animals, plants, and bacteria. And, as you might anticipate,
the bacterial enzyme was the first to be studied in great detail.
By 1969, the polypeptides that make up the E. coli RNA
polymerase had been identified by SDS polyacrylamide gel
electrophoresis (SDS-PAGE) as described in Chapter 5.
Figure 6.1, lane 1, presents the results of an SDS-PAGE
separation of the subunits of the E. coli RNA polymerase
by Richard Burgess, Andrew Travers, and their colleagues.
This enzyme preparation contained two very large subunits: beta (b) and beta-prime (b9), with molecular masses
of 150 and 160 kD, respectively. These two subunits were
not well separated in this experiment, but they were clearly
distinguished in subsequent studies. The other RNA polymerase subunits visible on this gel are called sigma (s) and
alpha (a), with molecular masses of 70 and 40 kD, respectively. Another subunit, omega (v), with a molecular mass
of 10 kDa is not detectable here, but was clearly visible in
urea gel electrophoresis experiments performed on this
same enzyme preparation. In contrast to the other subunits, the v-subunit is not required for cell viability, nor for
enzyme activity in vitro. It seems to play a role, though not
2
3
4
5
⫺
1
␤⬘
*
␶
␴
0.1% SDS GELS
␤
⫹
␣
Figure 6.1 Separation of s-factor from core E. coli RNA polymerase
by phosphocellulose chromatography. Burgess, Travers, and
colleagues subjected RNA polymerase holoenzyme to phosphocellulose
chromatography, which yielded three peaks of protein: A, B, and C.
Then they performed SDS-PAGE on the holoenzyme (lane 1), peaks A,
B, and C (lanes 2–4, respectively), and purified s (lane 5). Peak A
contained s, along with some contaminants (the most prominent of
which is marked with an asterisk), B contained the holoenzyme, and
C contained the functional core polymerase (subunits a, b, and b9).
(Source: Burgess et al., “Factor Stimulating Transcription by RNA Polymerase.”
Nature 221 (4 January 1969) p. 44, fig. 3. © Macmillan Magazines Ltd.
Table 6.1
Ability of Core and Holoenzyme
to Transcribe DNAs
Relative
Transcription Activity
DNA Template
Core
Holoenzyme
T4 (native, intact)
Calf thymus (native, nicked)
0.5
14.2
33.0
32.8
a vital one, in enzyme assembly. The polypeptide marked
with an asterisk was a contaminant. Thus, the subunit content of an RNA polymerase holoenzyme is b9, b, s, a2, v;
in other words, two molecules of a and one of all the others
are present.
When Burgess, Travers, and colleagues subjected the
RNA polymerase holoenzyme to cation exchange chromatography (Chapter 5) using a phosphocellulose resin, they
detected three peaks of protein, which they labeled A, B, and
C. When they performed SDS-PAGE analysis of each of these
peaks, they discovered that they had separated the s-subunit
from the remainder of the enzyme, called the core polymerase. Figure 6.1, lane 2 shows the composition of peak A,
which contained the s-subunit, along with a prominent contaminating polypeptide and perhaps a bit of b9. Lane 3
shows the polypeptides in peak B, which contained the
holoenzyme. Lane 4 shows the composition of peak C, containing the core polymerase, which clearly lacks the
s-subunit. Further purification of the s-subunit yielded the
preparation in lane 5, which was free of most contamination.
Next, the investigators tested the RNA polymerase activities of the two separated components of the enzyme: the
core polymerase and the s-factor. Table 6.1 shows that this
separation had caused a profound change in the enzyme’s
activity. Whereas the holoenzyme could transcribe intact
phage T4 DNA in vitro quite actively, the core enzyme had
little ability to do this. On the other hand, core polymerase
retained its basic RNA polymerizing function because it
could still transcribe highly nicked templates (DNAs with
single-stranded breaks) very well. (As we will see, transcription of nicked DNA is a laboratory artifact and has no
biological significance.)
Sigma (s) as a Specificity Factor
Adding s back to the core reconstituted the enzyme’s
ability to transcribe unnicked T4 DNA. Even more
significantly, Ekkehard Bautz and colleagues showed that
the holoenzyme transcribed only a certain class of T4 genes
(called immediate early genes), but the core showed no
such specificity.
Not only is the core enzyme indiscriminate about the T4
genes it transcribes, it also transcribes both DNA strands.
Bautz and colleagues demonstrated this by hybridizing the
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