Download Lecture 11 - Class I and Class III Factors

Eukaryotic transcription factors:
Class I and class III.
Class I promoters
•Service rRNA precursor gene
•Sequences are variable from one species to another
•Two critical regions:
core element, at the start of transcription, between -45 and
+20
upstream promoter element (UPE), between positions –156
and –107.
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Class I promoter elements
Linker scanning was used to identify important regions,
tested using in vitro transcription
Class I factors
Much simpler than class II
Preinitiation complex is much simpler:
•Polymerase I
•Two transcription factors:
~ SL1 in humans and TIF-IB in other organisms –
core binding factors
~ upstream-binding factor, UBF (mammals) or
upstream activating factor, UAF (yeast) –
assembly factors
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SL1 factor
Discovered in 1985 from HeLa cells.
Is a species-specific factor. Species-specificity of SL1 TF: it
could distinguish mouse and human rRNA template.
Necessary and sufficient to recruit polymerase.
SL1 also determines species specificity, as does the core
promoter element.
UPE-binding factor
UBF
•UBF – a transcription factor that stimulates transcription by
polymerase I; composed of two polypeptides, 94 and 97 kD
•97 kD alone is sufficient for UBF activity
•UBF and SL1 act synergistically to stimulate transcription.
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Activation of transcription from rRNA promoter by SL1 and
UBF
S1 assay was used to
measure transcription
from human rRNA
promoter in the presence
of RNA polymerase I and
various combinations of
UBF and SL1.
Conclusion: SL1 was required
for at least basal activity, but
UBF enhanced this activity.
They act synergistically.
Structure and function of SL1
TBP is essential for class I transcription.
How?
Tjian et al, 1992: SL1 is composed of TBP and 3 TAFs
These are: TAFI110, TAFI63, TAFI48.
Fully functional and species-specific SL1 can be
reconstituted from those components.
Binding of TBP to TAFIs precludes binding of it to TAFIIs.
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Class III factors
1980, Roeder et al discovered TFIIIA, that was bound to 5S
rRNA gene.
Roeder et al discovered that TFIIIA was needed for 5S rRNA
gene transcription, but not for the tRNA gene transcription.
1982 - Other factors: TFIIIB, TFIIIC were separated, they are
necessary for the III class genes.
5SrRNA – needs TFIIIA+TFIIIB+TFIIIC
Class III promoters
RNA polymerase III genes:
Classical – 5SrRNA, AV RNA
Non-classical (recently discovered) – U6 snRNA, EpsteinBarr virus EBER2 gene – resemble class II promoters.
Feature of class III – located within the genes themselves!
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‘Classical’ Class III genes with internal promoters
Findings of Brown et al, works with X. laevis 5SrRNA
TFIIIA
Binds DNA, has zinc-finger domain – finger shaped domain
that has aa that bind single Zn ion.
In TFIIIA the 4 aa are cysteines, 2 are histidines.
Has 9 Zn-fingers, it inserts them into the major groove at the
region of promoter – tight DNA-protein complex is formed.
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TFIIIB and TFIIIC
Classical class III factors require TFIIIB and TFIIIC.
Transcription of 5S rRNA gene depends upon TFIIIA, TFIIIB
and TFIIIC:
TFIIIC and A bind to internal promoter and help TFIIIB to bind
to the region upstream the transcription start. TFIIIB remains
bound and helps with the next rounds of transcription.
Role of TBP in the class III transcription
TFIIIC plays ‘organizing’ role as TBP to TATA-containing pol II
genes
?TBP is required for tRNA and 5S rRNA genes?
•TFIIIC binds to internal promoter’s A and B blocks.
•Promotes binding of TFIIIB, with its TPB to the region
upstream of transcription start site.
•TFIIIB promotes binding of polymerase at the start site.
•Transcription begins. When polymerase moves forward
making RNA, it possibly removes TFIIIC from the promoter.
•TFIIIB remains in place, ready to assist in anew round of
polymerase binding and transcription.
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Role of TBP in the
class III transcription
Model of preinitiation complex formation on TATA-less
promoters recognized by all three types of polymerases
1) Assembly factor binds first (UBP, Sp1 and TFIIIC in class
I, II and III promoters, respectively)
2) This attracts another factor that contains TBP (these are
SL1, TFIID or TFIIIB in class I, II and III promoters,
respectively).
3) These complexes are sufficient to recruit polymerase in
class I and III
4) In class II more GTFs are required.
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Model of preinitiation complex formation on
TATA-less promoters recognized by all three
types of polymerases
1) Assembly factor binds first
(UBP, Sp1 and TFIIIC in
class I, II and III promoters,
respectively)
2) This attracts another factor
that contains TBP (these
are SL1, TFIID or TFIIIB in
class I, II and III promoters,
respectively).
3) These complexes are
sufficient to recruit
polymerase in class I and III
4) In class II more GTFs are
required.
The assembly of preinitiation complex begins with
attachment of an assembly factor to promoter.
TBP plays important role in assembly.
Even if it is not the first bound factor, it becomes a part of
growing complex.
The specificity of TBP –which kind of promoter it binds
depends upon TAFs.
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Reading:
Class I and class III factors, pages 327-339.
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