Distinct roles of transcription factors

TFIIIB and TFIIIC in RNA polymerase

III transcription reinitiation
Ferrari et al. 10.1073/pnas.0403851101.

Supporting Information
Files in this Data Supplement:
Supporting Materials and Methods
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9

Supporting Figure 6

Fig. 6. Comparison of the U6/tDNA transcript ratios observed in the

experiment in Fig. 2D with those predicted by a stochastic reinitiation
model. The plot reports the U6/tDNA transcript ratios in function of the
number of cycles of U6 gene transcription. Filled circles with solid line,
experimentally observed ratios (experiment in Fig. 2 D); solid line,
theoretically predicted ratios with a 10% occupancy; dashed line,
theoretically predicted ratios with a 25% occupancy; and dotted line,
theoretically predicted ratios with a 50% occupancy.

Supporting Figure 7

Fig. 7. Low transcriptional activity of all-rTFIIIB. PICs were assembled

for 30 min on the I(TAT)LR1 tDNA, coding for a tRNA Ile(UAU) (lanes 1-7),
or on the tDNAGly(TCC) used throughout this study (lanes 8-14), by
incubating the template DNAs with TFIIIC fraction, rTBP, and rBrf1
proteins produced in Escherichia coli, and either crude B'' fraction (lanes
1, 2, 8,and 9) or various amounts of pure rBdp1 produced in insect cells
(lanes 3-7: 1, 3, 8, 25, and 100 ng, respectively; lanes 11-14: 100, 200,
400, and 800 ng, respectively; and lane 10, no addition). Pol III and the
four NTPs were then added and the incubation was continued for 30
min. The amount of B'' fraction used in lanes 1-2 and 8-9 provided no
more than 20 ng of Bdp1 polypeptide (see Materials and Methods).

Supporting Figure 8

Fig. 8. First-round initiation and elongation on the SCR1 gene. (A)

Stable PICs were assembled on the U6-SCR1 template for 20 min either
in the absence (–TFIIIC) or in the presence (+TFIIIC) of TFIIIC. A limiting
amount of Pol III (10 ng) was then added together with an NTP mixture
lacking CTP. After the indicated time periods, the missing nucleotide
was added in association with heparin, to allow for the completion of
the transcription cycle by heparin-resistant complexes. ( B) Stable PICs
were assembled on the U6-SCR1 template for 20 min either in the
presence (Left) or in the absence (Right) of TFIIIC. Pol III (10 ng) was then
added together with an NTP mixture lacking CTP and the incubation was
continued for 10 min. Elongation was resumed by the addition CTP, and
samples of the elongation reaction mixture were stopped at the
indicated times.

Supporting Figure 9

Fig. 9. Versatility of TFIIIC in promoting reinitiation on long

transcription units. The data in Figs. 4 and 5 show that TFIIIC similarly
stimulates reinitiation on the 520-bp-long SNR6_520 and SCR1
templates, despite their very different promoter architectures. As
schematically shown, on the SCR1 gene, the B block lies 52 bp
downstream of the transcription start site, with the terminator 467 bp
further downstream: thus, the TFIIIC-binding site is relatively close to
the transcription start site. On the SNR6_520 template, the terminator
lies 520 bp from the start site, and the B block lies ≈130 bp further
downstream: the main TFIIIC-binding site is thus >600 bp far away from
the transcription start site and from the upstream-assembled TFIIIB
complex. To explain the fact that TFIIIC stimulates reinitiation in the
context of these very different transcription complex geometries, it
must be assumed that this large factor can influence DNA compaction
both downstream and upstream of its main binding site. As speculated
in Fig. 9, such a compaction might involve DNA looping of large regions
of both templates. In the case of SNR6_520, looping would be favored
by simultaneous interaction of TFIIIC with the A and B blocks. In the case
of SCR1, an interaction of TFIIIC with a downstream region (such as the
terminator region, see ref. 1) would be required to loop out the DNA
between the B block and the termination site, thus reducing the distance
between the terminating polymerase and the promoter complex. Other
forms of TFIIIC-mediated DNA compaction, involving for example
TFIIIC-Pol III interactions and/or DNA wrapping around these proteins,
cannot be excluded. To explain the reinitiation role of TFIIIC, it should
also be assumed that this factor does not undergo dissociation from the
template upon the passage of elongating Pol III. Such an assumption
contrasts with the reported displacement of TFIIIC by elongating Pol III
(2). In that case, however, transcription was carried out in the absence of
TFIIIB, which is known to stabilize the TFIIIC-DNA interactions (3) and
may thus be required for TFIIIC retention on the template during
transcription. The hypothesis that TFIIIC is removed from its binding
sites by the elongating polymerase is also supported by the outcomes of
genome-wide studies of the localization of Pol III transcription
components based on chromatin immunoprecipitation (4-6). In
particular, a dramatic increase in TFIIIC occupancy at class III loci has
been observed during acute repression of class III gene transcription,
concomitant with a strong decrease in Pol III association, as if the
occupancy of class III genes by TFIIIC and Pol III were mutually exclusive
(5). Our observation that TFIIIC is required for reinitiation, at least on
some genes, can be reconciled with these observations by admitting
that Pol III-displaced TFIIIC may remain tethered to the transcription
complex, for example, through interactions with TFIIIB. Alternatively, it
is possible that the apparent lesser occupancy by TFIIIC on actively
transcribed genes, revealed by chromatin immunoprecipitation, reflects
a reduced accessibility of TFIIIC to antibodies, due to Pol III
encumbrance.