,
Direct imaging of E. coli DNA transcription
process.
J. Usukura#, H.Tagami$, H. Aiba$
#. Department of Anatomy, Nagoya University Nagoya ,466 Japan
$. Department of Molecular Biology, Nagoya University Nagoya, 466 Japan
Purpose: To visualize DNA transcription process at molecular level.
Methods:
Transcription process was carried out in vitro using E. coli RNA
polymerase holoenzyme (RNAP) and lkb DNA template containing lac UV 5
promoter. Specific and non-specific RNAP / DNA complexes were formed with or
without 20 pg/pl heparin respectively. Elongation complex synthesizing mRNA
was transformed by adding 4 NTPs to specific (open) complex , but concentration
of UTP was l/10 of the other NTPs in order to cease the synthesis of mRNA at
appropriate length. Assembled samples were observed while tilting them under
electron microscopy equipped with newly developed energy filter (1) combined
with improved low angle rotary shadowing (2) and ice embedding methods.
Closed (non-specific), open and elongation complexes
Results and Discussion:
of RNAP and DNA in transcription process are analysed morphologically by
stereoscopic imaging. In this study, energy filter enable us to analyse structure of
optimal shadowed DNALRNAP complexes by contrast enhancement effect as well as
elemental mapping of phosphate along the DNA strand. The rotary shadowed RNAP
consisted clearly of two large blobs (presumed to be p,p’ subunits) stacked on two
small blobs (presumed to be 2a subunits), forming large channel between them, 3
nm in width. Total appearance was similar to the RNAP model proposed by
Polyakov et aL(3). On both nonspecific and specific binding with the promoter
region, DNA strand was found in this large channel. Although nonspecific bindings
dissociated on applying heparin, specific binding (open complex) was remained
there while kinking DNA in the flank. We succeeded in the direct visualization of
mRNA elongating from template DNA. Newly synthesizing mRNA seemed to be
coming out from the 3’ gate of large channel between a and p subunits.
References:
J. Electron Microsc., 45 (1996 ) 307-3 13
Taya, S.et al.
Hirako, Y.et al. J. Biol. Chem., 271 (1996) 13739-13745
Polyalov,A. et al. Cell 83 (1995) 365-373
Fig. 1 Stereoscopic pair of nonspecific binding . Each RNAP bind
with DNA at different angle.
Scale bar; 25 nm
Fig. 2 Stereoscopic high power
view of open complex. It is clear
that a DNA strand is passing
through the central large channel of
kNAP. Scale bar: 25 nm
Fig. 3 Low magnification image of elongation complex showing synthesizing mRNA ( arrow).
Promoter was inserted in a center region of template DNA, a mRNA was synthesized from right to
left in this figure. Scale bar; 25 nm
Fig. 4 Stereoscopic high power
view of a part of above figure. It is
obviously observed that mRNA is
coming out from 3’ gate of the
RNAP central channel, then crossing
beneath the template DNA in this
figure. Scale bar; 25 nm
p+
A p
p&i/