Download No Slide Title

The Yeast nRNAP II
• Has 12 subunits, based on traditional enzyme
purification and epitope tagging.
• Gene knockouts indicate that 10 subunits are
essential, & 2 are required under certain
conditions (4 and 9).
• The 2 large subunits (genes Rpb1 and Rpb2)
have regions of homology with the b and
b’ subunits of the E. coli RNAP and seem
to function similarly.
• RPB1 is responsible for a-aminitin sensitivity.
Epitope tagging
1. Add “tag” (small peptide) to
a subunit gene.
2. Transform in tagged gene.
3. Use a specific antibody for
the tag to “immunoselect”
the tagged protein.
4. Other proteins that come
down with the tagged
protein are detected by
SDS-polyacrylamide gel
electrophoresis.
Fig. 10.6
RNAP II purified
using the
epitope tag on
Rpb3; has 10
subunits.
RNAP II
purified from
wild-type
yeast: 12
subunits.
Rpb1 subunit is
phosphorylated.
1992
1990
Fig. 10.7
nRNAP II Heterogeneity
• Largest subunit - Subunit II (Rpb1 gene) is
phosphorylated on carboxyl-terminal domain
(CTD).
• 3 forms of this subunit
IIa - primary product
IIb - derived from IIa by removal of CTD
(artifact of purification)
IIo - phosphorylated form of IIa
• Functionally different: IIa-enzyme binds
promoter; IIo-enzyme is in elongation phase.
Structure of yeast RNAP II lacking Rbp4 and 7
Fig. 10.10
Surface structure of yeast RNAP II
Mg2+ at active site - pink sphere
Fig. 10.11
Model for the path of a straight DNA
in the initiation complex
Clamp opens to let DNA template have access.
Fig. 10.12
Structure of the elongation complex.
Figure 10.13
Fig.
10.14
Figure 10.14
Synthetic DNA used to obtain a mimic
of a transcribing complex after
translocation
Dideoxynucleotided
Fig 10.17
Fig.
Matched and Mismatched Nucleotides in A (active) and
10.18
E (entry) sites
Fig 10.18
Promoters for the 3 Nuclear
RNA polymerases (nRNAPs)
Order of topics:
1. Class II promoters (for nRNAP II)
2. Class I promoters (for nRNAP I)
3. Class III promoters (for nRNAP III)
4. Enhancers and Silencers
Figure 10.20
Generic Class II Promoter
1.
2.
3.
4.
5.
Upstream element
TFIIB recognition element
TATA Box (at approx. –25)
Initiator
Downstream promoter element (can substitute for
TATA box)
1.
2.
3.
4.
Promoters may lack one or more of these and still function.
5.
TATA Box of Class II Promoters
• TATA box = TATAAAA
• Defines where transcription starts.
• Also required for efficient transcription for
some promoters.
• Some class II promoters don’t have a
TATA box.
Effects of Deletions in the SV40 Early Promoter
on the Transcription Start Site
(b) Mapping of the transcription start site on the
mutant DNAs by S1 nuclease digestion.
Fig 10.21
extension.
S1 mapping of the 5’ end of a RNA Transcript
A 5’ end-labeled single-stranded DNA probe is prepared from the
template strand. After hybridization to RNA and digestion with S1,
the size of the protected DNA indicates approx. how far to the RNA
5’-end.
Similar to Fig. 5.26
High resolution analysis of
the 5’-end of an RNA
transcript by primer
extension.
Primer is an end-labeled DNA
oligonucleotide (~20 nt) that is
complementary to a sequence
in the RNA ~150 nt from the
expected 5’ end.
Lane E- extended DNA product
Lanes A,C, G, T – sequence
ladder generated with the same
oligo primer, but on the
corresponding cloned DNA.
From Fig. 5.29
Linker scanning mutagenesis of a stretch of DNA.
Replace ~10 bp of natural sequence with 10 bp of
synthetic DNA.
Do this periodically throughout the stretch of DNA you
want to examine for important sequences.
From Fig. 10.22
Linker scanning
mutagenesis of the
Herpes virus tk
promoter identifies
2 important
upstream regions.
DNA was injected into
frog oocytes, and the
transcribed RNA
analyzed by primer
extension.
Regions -47 to -61 and
-80 to -105 contain GC
boxes (GGGCGG and
CCGCCC).
Fig. 10.23
Upstream Elements of Class II
• Can be several of these, two that are often found:
1. GC boxes (GGGCGG and CCGCCCC)
– Stimulate transcription in either orientation
– May be multiple copies
– Must be close to TATA box (different from enhancers)
– Bind the Sp1 factor
2. CCAAT box
– Stimulates transcription
– Binds CCAAT-binding transcription factor (CTF) or
CCAAT/enhancer-binding protein (C/EBP)
Class I Promoters (for nRNAP I)
• Sequences less conserved than Class II
• Usually 2 parts:
– UCE : upstream control element , -150 to -100 in
human rRNA
– Core: from - 45 to +20
• Spacing between elements also important
-150
-100
UCE
-50
+20
Core
Results of Linker Scanning mutagenesis of the human
rRNA promoter.
The DNA was transcribed in vitro, and the efficiency is
expressed relative to the wild-type promoter.
Fig. 10.24
Class III promoters (for nRNAP III)
•
1.
2.
3 types:
Two (Types I and II) have Internal promoters
- 5S rRNA (Box A, Intermediate element, Box C)
- tRNA (Box A, B)
Type III is upstream of coding region
- e.g., human U6 RNA
- contain TATA box and resemble class II promoters
Effect of deletions at the 5’-end
of the Xenopus 5S rRNA gene
on its transcription in vitro.
Result: It required deleting more
than 50 bp into this small gene to
knock out its transcription.
Conclusion: the promoter for
the 5S rRNA gene is internal!
Numbers at bottom of lanes are the bp deleted.
Fig. 10.27
Enhancers and Silencers
• Enhancers stimulate transcription, silencers
inhibit
• Both are orientation independent
– Flip 180 degrees, still work
• Both are position independent
– Can work at a distance from promoter
• Bind regulated (tissue-specific) transcription
factors
An enhancer in an intron of the γ2b gamma-globulin
Fig. 10.30
gene.
(a) Genes were constructed with the enhancer inverted (B), or moved
upstream of the gene (C) and inverted (D). The DNAs were transfected
into mouse cells and synthesis of the protein was assessed by pulselabeling with a radioactive amino acid and immunoprecipitation.