Promoters for the 3 nuclear RNA polymerases nRNAPs

1
Promoters for the 3 nuclear RNA polymerases
(nRNAPs)

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

2
RNAP II Promoters (a.k.a. Class II)

• Class-II promoters usually have 4 components
• Upstream element
• TATA Box (at approx. 25)
• Initiation region
• Downstream element

1. 2. 3. 4.
Many class II promoters lack 3 and 4.
3
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 (e.g., for housekeeping
  genes or some developmentally regulated genes
  (e.g., homeotic)) dont have a TATA box.

4
Transcription starts at a purine 25-30 bp from
the TATA box.
Normal promoter.
SV40 early promoter analyzed in vivo.
Fig. 10.28
5
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 probe tells approx. where
transcription started.
From Fig. 5.27
6
High resolution analysis of the 5end 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. 5.30
7
TATA box also important for transcription
efficiency for some promoters.
Rabbit globin promoter, tested in Hela cells, and
assayed by S1 mapping of transcript 5 end.
Fig. 10.29
8
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. 10.30
9
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. 10.31
10
Upstream Elements of Class II

• Can be several of these
• Two that are found in many class II promoters
• 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)

11
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
12
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. 10.32
13
Class III promoters (for nRNAP III)

• 2 types
• Internal promoters
• - 5S rRNA (Box A, Intermediate element, Box C)
• - tRNA (Box A, B)
• Class II like promoters
• - contain TATA box
• - 7SL gene, promoter is upstream of coding region

14
Fig. 10.34
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 knockout its transcription.
Conclusion the promoter for the 5S rRNA gene is
internal!
Numbers at bottom of lanes are the bp deleted.
15
Enhancers and Silencers

• Enhancers stimulate transcription, silencers
  inhibit.
• Both are orientation independent.
• Flip 180 degrees, no effect
• Both are position independent.
• Can work at a distance from promoter
• Enhancers have been found all over
• Bind regulated transcription factors.

16
An enhancer in an intron of a gamma-globulin gene.
Fig. 10.40
(a) Genes were constructed with the enhancer
inverted (B), with it 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, immunoprecipitation, and separation
by SDS-PAGE and autoradiography.