The Yeast nRNAP II

1
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.

2
Epitope tagging

• Add tag (small peptide) to a subunit gene.
• Transform in tagged gene.
• Use a specific antibody for the tag to
  immunoselect the tagged protein.
• Other proteins that come down with the tagged
  protein are detected by SDS-polyacrylamide gel
  electrophoresis.

Fig. 10.6
3
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
Fig. 10.7
1990
4
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.

5
Fig. 10.10
Structure of yeast RNAP II lacking Rbp4 and 7
6
Fig. 10.11
Surface structure of yeast RNAP II
Mg2 at active site - pink sphere
7
Fig. 10.14
Model for the path of a straight DNA in the
initiation complex
Clamp opens to let DNA template have access.
8
Fig. 10.15a,bNucleic acid- RNAP interactions
in the elongation complex
9
Figure 10.16
10
Figure 10.18
The Transcription Bubble
11
Promoters for the 3 nuclear RNA polymerases
(nRNAPs)

• Order of topics
• Class II promoters (for nRNAP II)
• Class I promoters (for nRNAP I)
• Class III promoters (for nRNAP III)
• Enhancers and Silencers

12
Generic Class II Promoter
Figure 10.20

• Upstream element
• TFIIB recognition element
• TATA Box (at approx. 25)
• Initiator
• Downstream promoter element (can substitute for
  TATA box)

1. 2. 3. 4.
5.
Promoters may lack one or more of these and still
function.
13
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 dont have a TATA box.

14
Transcription starts at a purine 25-30 bp from
the TATA box.
Normal promoter.
Deletions of the SV40 early promoter Analyzed
by S1 mapping of RNA 5 ends in vivo.
15
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
16
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
17
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
18
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
19
Upstream Elements of Class II

• Can be several of these, two that are often
  found
• 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
• CCAAT box
• Stimulates transcription
• Binds CCAAT-binding transcription factor (CTF) or
  CCAAT/enhancer-binding protein (C/EBP)

20
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 -50 20
UCE
Core
21
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
22
Class III promoters (for nRNAP III)

• 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

23
Fig. 10.27
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.
24
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
• Enhancers have been found all over
• Bind regulated transcription factors

25
An enhancer in an intron of the ?2b
gamma-globulin gene.
Fig. 10.32
(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 pulse-labeling with a radioactive
amino acid and immunoprecipitation.