Download 1 Transcription in eukaryotes Eukaryotic RNA polymerases

Transcription in eukaryotes
Eukaryotic RNA polymerases
Separation of the three nuclear polymerases
RNA polymerase I – nucleolar rRNA synthesizing enzyme.
RNA polymerase II - nucleoplasmic enzyme.
RNA polymerase III – nucleoplasmic enzyme.
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Roles of RNA polymerases
In studies RNA polymerases were used to transcribe various
genes.
Purified polymerases were shown to transcribe certain genes,
but not others in vitro.
RNA polymerase I – makes rRNA
RNA polymerase II – makes heterogeneous nuclear RNA
(hnRNAs)
and small nuclear RNA (snRNAs)
hnRNAs – precursors of mRNAs
snRNAs – needed for maturation of hnRNA to mRNA
Polymerase III - makes precursors of tRNAs, 5S rRNAs and
some small RNAs: U6 snRNA – participates in splicing, 7SL
RNA – small RNA involved in signal recognition in the
synthesis of secreted proteins, 7SK RNA – small RNA of
unknown function, the adenovirus VA (virus-associated
RNA), and the Epstein-Barr virus EBER2 RNA.
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Polymerase subunits
The first subunit structures for eukaryotic RNA polymerase II
were reported by 2 groups.
Yeast model:
Genes for all subunits were cloned and sequenced.
Names of subunits - Rpb1-Rpb12, genes RPB1-RPB12.
RPB – RNA polymerase B (not II).
NB:
Similarities – each
contains large subunits
and a variety of smaller
ones
These structures
resemble that of the
prokaryotic core
polymerases, which
contain high-mass
subunits and a low-mass
subunits (α2).
Yeast RNA polymerases
have 5 common subunits
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RNA polymerase II
Problem – enzyme is very complex, difficult to say which
polypeptides that co-purify with the polymerase activity are really
subunits or contaminants.
Two approaches to study:
•Separate subunits and then reconstitute to structure and check
the function
But: It was not yet possible to reconstitute RNA polymerase
from separate subunits
•Another option: find genes for all putative subunits, mutate them
and look for the function.
All the genes were discovered, cloned and sequenced. They
clone for 12 putative subunits of yeast polymerase II.
Each of polypeptides is encoded in a single gene in the yeast
genome.
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The epitope tagging
•Antibodies against epitope tag
allowed immunoprecipitation of the
whole polymerase
•Addition of strong detergent SDS
leads to separation of subunits
•They can be detected by
electrophoresis of denatured
subunits
The epitope tagging
Yeast RNA Pol II
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Human RNA pol II subunits (Table 10.2)
Rpb1 - ortholog of β’, binds DNA, involved in start selection
Rpb2 - ortholog of β, contains active site,
involved in start site selection, elongation rate
Rpb3 – ortholog of α, may function with Rpb11
Rbp4 – subcomplex with Rpb7, stress response
Rpb 5 – shared with PolI, II, III, target for activators
Rpb 6 - shared with PolI, II, III, functions in assembly and
stability
Rpb7 – Subcomplex with Rpb4
Rpb 8 - shared with PolI, II, III, oligonucleotide binding
domain
Rpb9 – Zinc ribbon motif involved in elongation, functions in
start site selection
Rpb 10- shared with PolI, II, III
Rpb 11- May function with Rpb3 as ortholog of α
Rpb 12- ortholog of α
Response of the PolΔ4/7 to a yeast promoter
Kornberg et al: transcribed a
construct containing yeast CYC1
promoter and a GAL4 binding
site in a cell-free extract of rpb-4
cells. They supplement ed the
reactions with either 4 or 7
subunits or GAL4-VP16 factor (or
both).
PolIIΔ4/7 was unable to
recognize promoter (lane 1), with
the help of transcription factor it
could manage a little
transcription (lane 2)
The mt polymerase
supplemented with 4 and 7
subunits could recognize
promoter even in the absence of
transcription factor.
Mt polymerase with 4/7 and TF
– transcription is very active.
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Source:
Molecular Biology, Weaver, 2005
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