ABSTRACT
We have studied the structure of recombinant rat UBF (rrUBF), an RNA polymerase
I transcription factor, by electron microscopy and image analysis of single
particles contrasted with methylamine tungstate. Recombinant rat UBF appeared
to be a flat, U-shaped protein with a central region of low density. In the dominant
projections, 2-fold mirror symmetry was seen, consistent with the dimerization properties
of this molecule, and of dimensions in agreement with the length of DNA that
rat UBF protects in footprinting studies. Electron microscopy of various rrUBF-DNA complexes confirmed that our recombinant protein was fully able to
bind the 45S rDNA promoter, and that it caused substantial bends in the DNA.
Upon extended incubation in a droplet covered by a lipid monolayer at the
liquid-air interface, rrUBF formed long filamentous arrays with a railway track
appearance. This structure was interpreted to consist of overlapping rrUBF
dimers 3.5 nm apart, which value would represent the thickness of the protein.
Our results show rrUBF to interact with and bend the promoter DNA into a
roughly 10 nm diameter superhelix. Based on all these electron microscopical results, an
atomic structure was predicted by homology modelling of the HMG fingers, and
connected by energy minimized intervening segments.
The synthesis of ribosomal RNA (rRNA) in vertebrates depends on the efficient
initiation of rRNA gene transcription by RNA polymerase I and its associated
transcription factors (
1
-
3
). Before RNA polymerase I can bind to the promoter region, there must be
interactions amongst a species specific factor SL1 containing the TATA box
binding protein (TBP), along with other TBP-associated factors (TAFs), the upstream binding factor (UBF), the core
promoter element (CPE), and the upstream promoter element (UPE) (
1
-
8
). UBF has been implicated in the recognition of the promoter region, binding
both to the CPE and UPE. An extended DNase I footprint occurs on the rRNA
promoter when UBF and SL1 are present together, implying that there is a
cooperation between UBF and the binding of one or two complexes of SL1 (
7
-
11
). In some cases UBF is a requirement for transcription initiation but instances
where transcription initiation can occur without UBF have been described (
8
,
12
). One model of 45S rRNA transcription initiation has the UBF dimer binding to
the upstream promoter element and bending the DNA further, bringing the CPE and
UPE into closer proximity (
13
,
14
). [The DNA is already intrinsically bent in this region (
15
).] Subsequently, SL1, RNA polymerase I, and ancillary transcription factors
such as factor 1c, the core promoter binding factor (CPBF), and factors E1BF/KU
all bind, resulting in formation of a stable transcription initiation complex
with potential subsequent nucleosomal disruption (
16
). The role of UBF in the committed complex has yet to be precisely defined (
17
).
The cDNAs for UBF have been cloned from a variety of species including human,
mouse, rat and
Xenopus laevis
(
18
-
21
). The high sequence conservation amongst these species suggests that there is a
large degree of structural similarity between the different proteins. There are
three distinct regions to UBF. The N-terminal region, consisting of the first 100 or so amino acids, makes up
the dimerization domain which is similar to the helix-turn-helix dimerization domains found in RNA polymerase II
transcription factors (
2
2
). Approximately half of the mammalian UBF sequence consists of four tandem
repeats of an 80 amino acid segment. They are the highly basic DNA binding
domain referred to as the HMG-box. This region has high homology to the DNA binding domain in the non-histone chromatin associated protein HMG1 (
18
). Finally, at the C-terminal end of the UBF protein is a highly acidic region which is
involved in transcription initiation, but which is not required for DNA binding
or nucleolar localization (
23
-
25
). The C-terminal tail also contains serine residues that can be phosphorylated by
casein kinase II. Unphosphorylated UBF cannot stimulate transcription, and
additional cellular protein kinases are required for growth-dependent UBF phosphorylation (
26
,
27
). Purified rat, mouse and human UBF fractions contain two proteins, of 97 and
95 kDa, designated UBF1 and UBF2, respectively. UBF2 has a 37 amino acid
deletion in the second of the four HMG-boxes (
20
). Crosslinking studies have shown that rat UBF (rUBF) exists as a dimer in
solution (
23
), with the N-terminal region and HMG-box 2 being important for dimerization. Based on NMR spectroscopy,
the solution structure of an HMG-box can be described as an arrowhead formed from three alpha helices,
representing a novel DNA binding domain (
24
,
25
).
Footprinting studies and Southwestern blots have identified the HMG-boxes closest to the N-terminal end as being the dominant DNA binding domains, with the
first HMG-box being the most significant to specific DNA binding (
18
,
22
-
24
). These N-terminal HMG-boxes are also more closely related to the original HMG1 motif. The
HMG-boxes alone can bind non-specifically, but preferentially to four-way junction DNA (
28
). The C-terminal HMG-boxes and the acidic tail are required for the extended footprint on
the rDNA gene promoter in the presence of SL1, implicating them in protein-protein contact between UBF and SL1 (
10
). HMG-boxes from lymphoid enhancer factor 1, SRY (a transcription factor encoded
by the sex-determining region of the human
In this paper, transmission electron microscopy and image analysis of
preparations of rrUBF as single particles and as filaments have yielded a model
of rrUBF structure to low resolution, and with electron spectroscopic images of
rrUBF-DNA complexes allowed definition of its interaction with the rRNA gene
promoter.
The mRNA for rat UBF1 was cloned, and an N-terminal His-tagged form without the acidic tail was overexpressed in
E.coli
. This His-tagged recombinant rrUBF was purified by affinity chromatography on a Ni
column according to the manufacturer's instructions (Qiagen). Proteins were
dialysed against a buffer containing 20 mM HEPES-KOH pH 7.9, 100 mM KCl, 5 mM MgCl
2
, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT and 0.5 mM PMSF (buffer A), and stored
at -70oC until use. The high degree of homogeneity of the rrUBF preparation
was confirmed by SDS-PAGE with both Coomassie Blue and silver staining.
Two plasmids were used, p2.0 and p8.5, both containing fragments of the rat 45S
rRNA gene promoter (Fig.
1
) (
35
,
36
). Plasmid p2.0 contains 170 bp of non-transcribed spacer (NTS) upstream of the initiation site, and 1829 bp of
the external transcribed spacer (ETS), cloned into pBluescript. Plasmid p8.5
contains 3686 bp of the NTS and 4812 bp of ETS and part of the 18S rRNA gene.
Plasmid p2.0 was purified by cesium chloride gradient centrifugation. A 2000 bp
Sal
I
fragment containing the rat 45S ribosomal RNA gene promoter was cut from this
plasmid, purified on a 1% agarose gels excised from the gel, and the DNA
extracted by electroelution and ethanol precipitated in a dry ice-ethanol bath. DNA was verified free of agarose by electron microscopy.
The p8.5 plasmid was used as the template from which to amplify, by the
polymerase chain reaction (PCR), a 445 bp region containing 230 bp of the NTS
and 215 bp of the ETS. The 445 bp PCR product was purified by high pressure
liquid chromatography.
Purified rrUBF at 120 [mu]g/ml was adsorbed to an ultrathin carbon film on a 400 mesh Cu or Ni grid
for 2 min in buffer A, or in a low salt buffer obtained by concentrating the
above sample through a 10 000 Da cutoff ultrafiltration device (Millipore
Ultrafree MC 10 000), and diluting with 20 mM HEPES-KOH pH 7.9, 0.5 mM EDTA and 10 mM KCl. Positive staining was then
attained by addition of 0.25% methylamine tungstate to the carbon film for 1
min, washing twice with double-distilled water (ddH
2
0), and air drying. The experiments were repeated with buffer alone as a
negative control. Samples were micrographed at a nominal magnification of 50
000* on a JEOL 100-CX operating at 80 kV. Each specimen area was not pre-illuminated prior to being micrographed, to minimize the
total electron dose.
Electron micrographs of positively stained molecules of rrUBF imaged in the
bright field mode were visually scanned to select distinct particles of
recurring appearance and size. Particles of size considerably greater or
smaller than that expected for rrUBF (~10 nm diameter) were considered to be aggregates or debris and were not
selected. Putative rrUBF particles were digitized and stored in arrays of 128
by 128 pixels (picture elements), where one pixel corresponded to an area of
0.125 nm by 0.125 nm at the object level. A total of 287 distinct particles
were selected.
Single particle analysis of digitized images was performed in the framework of
the IMAGIC image processing system as described elsewhere in detail (
37
-
41
), and summarized here. The single macromolecular images were pre-treated by band-pass filtering to suppress noise, normalized to have a common
variance, and put into register with one another by a multi-reference alignment. We used both multi-variate statistical and visual classification to subdivide this set
of 287 images into eight major classes according to criteria of size and shape.
Averaging was done using the 20 images that gave the highest correlation
coefficient with the reference image in each class. Four of these classes were
considered `best' in terms of homogeneity of constituents and the clarity of
detail in the final average. These averages of homogeneous subsets of aligned
images represented the two-dimensional constructions of particular recurring projections of the
macromolecular complex.
The 2000 bp
Sal
I
restriction fragment, with the 45S rRNA promoter positioned closer to one end,
was used to form rrUBF-DNA complexes. The binding reaction was performed at room temperature for
15 min in buffer A, at a ratio of two rrUBF molecules per DNA fragment (0.05 [mu]g/[mu]l DNA: 0.1 [mu]g/[mu]l rrUBF) in a total volume of 20 [mu]l. Following a 15 min incubation in the presence of 0.1%
glutaraldehyde, the complexes were spread for microscopy on ultrathin carbon
films either with the DMP-30 microdroplet technique (
42
) or directly onto carbon coated grids recently exposed to a tungsten light
source (
43
). Formation of complexes with 445 bp DNA was performed in the same manner,
except that 0.01% glutaraldehyde was used, along with a brief exposure (1 min)
to short-wave ultraviolet light (254 nm) at the end of the 15 min incubation.
Electron spectroscopic imaging (ESI) of unstained complexes was performed on a
Zeiss EM902 equipped with a Castaing-Henry energy filter at a nominal
magnification of 30 000*. ESI used scattered electrons with energy losses within a narrow range,
and was `tuned' to phosphorus by imaging at 150 +- 8 eV loss. These 150 eV loss images thus presented the DNA with high
contrast without the need for exogenous heavy metal stains or shadowing (
33
). All microscopy was performed with the specimen side up, facing the electron
source. Measurements of the position of binding of rrUBF, the complex size, and
the bend angle effected in the DNA by protein binding were performed on images
captured on an Alpha Innotech 1000 (Alpha Innotech Corp., San Leandro, CA) gel
scanner. A program was written in Microsoft Visual Basic, running under
Microsoft Windows, which allowed interactive delineation of the path of DNA or
the periphery of the complex using the mouse, and subsequent calculation of
distances, areas and bend angles. A total of 297 images of the rrUBF: 445 bp
DNA complexes was thus analysed.
A lipid monolayer approach was utilized to attempt to form planar arrays of
rrUBF (
44
-
46
). A drop of size 10 [mu]l of rrUBF at a concentration of 0.2 [mu]g/[mu]l was placed in a 1 mm deep by 3 mm diameter well in a block of
Teflon. Then <= 1 [mu]l 7:3 phosphatidylcholine-phosphatidylserine (Avanti Polar Lipids, Alabama) solution in
chloroform-hexane was touched to the surface to form a lipid monolayer at the liquid-air interface. The droplet was incubated in a humid chamber under
argon, at 4oC for times ranging from 30 min to overnight. Arrays were then picked up
onto ultrathin carbon coated 400 mesh Ni grids, contrasted with 2% uranyl
acetate, and observed at a nominal magnification of 50 000* as before.
With the known molecular weight of the rrUBF dimer, the average density of
proteins, the size and shape constraints of the rrUBF dimer determined by
electron image analysis of single particles and the appearance of the linear
filaments, and the length of the upstream binding element protected by UBF in
footprinting assays, we hypothesised that we could create a three-dimensional model of the surface of the rrUBF dimer as a first step
towards more detailed predictions. The symbolic mathematical language MAPLE V
was used in this task (
47
).
Recently, the structures of individual HMG-boxes from rat and hamster have been solved using solution NMR (
47
24,25). We obtained the coordinates of the HMG-box from one of these studies (
25
), and predicted the coordinates of the HMG-boxes within the rrUBF using multiple sequence alignment and homology
modelling functions of the INSIGHT II molecular modelling software package
(Biosym Corporation, Parsippany, NJ).
Our first approach to analysing the structure of rrUBF was bright-field transmission electron microscopy and single particle analysis of
highly purified preparations. Figure
2
shows both fields of view and enlargements of individual molecules of the rrUBF
spread on the thin carbon support, contrasted by basic methylamine tungstate
stain. The molecules were ~10 nm in diameter, and were well separated from each other. Observation of
the buffer alone (without protein) demonstrated a negligible amount of
particulate material due to precipitants in the buffer or stain. This negative
control discounted the possibility that these images were artefacts, and
indicated that the recurring motifs of the expected size range that were
observed were indeed rrUBF.
The enlargements in Figure
2
b show a number of the individual rrUBF molecules that were digitized and used
for image analysis. These samples demonstrate the typically observed recurring
views, some toroidal- or U-shaped, with a central depression and with a suggestion of 2-fold mirror symmetry. The independent averages of
characteristic projections obtained by image analysis are shown in Figure
3
. Two-fold symmetry is evident especially in Figures
3
a and b, consistent with rrUBF existing in dimeric form. The rrUBF dimers have
dimensions of approximately 8 * 6 nm
2
, and appear as a closed toroidal- or an open U-shaped molecule, with a central area of low density. Based on these
initial results, a surface model for rrUBF was generated using Maple V (
47
). This model was essentially a torus of internal radius
r
, bend radius
R
, truncated to 7/8 of a turn (Fig.
4
). The model shown here is the result of refinement using also the appearance of
the rrUBF filaments (below).
In order to verify that the purified rrUBF indeed bound DNA specifically at the
upstream promoter element, rrUBF was incubated with the 2000 bp fragment or 445
bp fragment of DNA and visualized. Figures
5
and
6
show several rrUBF-DNA complexes imaged electron spectroscopically having neither been
stained with a heavy metal salt nor rotary shadowed with platinum-palladium to increase contrast. The rrUBF molecule was usually seen to
bind the DNA fragment preferentially at one end. This observation suggests that
rrUBF is correctly bound to the promoter region. Particles ~10 nm in size are interpreted to represent the dimeric form of rrUBF, while
those structures roughly 20 nm in diameter or greater probably represent
multimeric rrUBF complexes. Frequently the DNA undergoes a sharp bend,
sometimes looping back on itself. This bend angle usually measures 90-180o but would actually be more if the plasmid DNA is fully wrapped
around the rrUBF molecule. The mean length of the 445 bp DNA complexed with
rrUBF was 129 +- 18 nm (+- standard deviation), and of naked DNA it was 149 +- 10 nm (Fig.
7
). The measured length of bound DNA was thus shorter than for naked DNA by
roughly 20 nm or 60 bp. A one tailed
t
-test for means shows this difference to be statistically significant at a
level >99%. Within the limits of measurement error, these values are equivalent
to the amount of DNA protected by UBF in footprinting studies, and to one
supercoil turn of DNA as described above. The complex diameter was found to lie
most often in the range 6-18 nm (Fig.
7
b). Those complexes >10 nm in size could represent higher-order polymerisation.
We have deduced a low resolution structure of the important 45S rDNA
transcription factor UBF from rat. In dimeric form, recombinant rat UBF is a
flat, toroidal-shaped protein which has the potential of being flexible. We have also
shown the rrUBF dimer to bind the 45S rDNA gene promoter, and to induce bends
into the DNA. The images are consistent with the protein interacting with the
DNA minor groove. We have also shown rrUBF to form filamentous arrays at high
concentrations on lipid monolayers. This observation suggests the rrUBF dimer
is capable of forming multimers, by an overlapping association between its
exterior surfaces. These results, and knowledge of the tandem repeat of the HMG-boxes in rrUBF, suggested a model of the rrUBF dimer which has the DNA of
the promoter region wrapped around the outer edge of the toroidal shape,
bringing the CPE and UPE in close proximity. This geometry would facilitate the
interaction of SL1 with rrUBF, and the binding of SL1 to the UPE and CPE, and
would be consistent with the extended footprint observed when human UBF and SL1
are footprinted on the human promoter. Moreover, this structure would be
consistent with the mechanism by which HMG proteins bind to DNA.
This work was supported in part by a grant from the Natural Sciences and
Engineering Research Council of Canada to G.H., and by National Institutes of
Health grants GM46991 and HL46738 to L.I.R. We are grateful to Mr Philip Chang
for assistance with the HMG modelling, Dr Aled Edwards for instruction and
advice on lipid monolayer crystallization, Mr Lutz Schmitt and Dr Robert Tampé for their interaction in the Ni-chelator lipid monolayer experiments, Mr Michael LaCroix for
writing the program for measurement of parameters on micrographs, and to Mr Yew-Meng Heng and Dr Peter Ottensmeyer for assistance with and use of their
Zeiss EM902 for electron spectroscopic imaging.
REFERENCES
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