Download Methods for Studying Eukaryotic Gene Control

Methods for Studying
Eukaryotic Gene Control
1. Multiple eukaryotic RNA polymerases
for rRNA, tRNA and mRNA synthesis
2. Methods for Mapping cis- regulatory
sequences: proximal and distal elements
3. Finding the Start of Transcription
4. Complex Cis-control Modules are Bound
by sequence specific factors to direct
temporal and spatial regulation
Assay for non-specific RNA polymerase activity
+ RNA polymerase
+ [a-32P]rNTPs/ Mg+2
Non-specific or synthetic
DNA fragments
*
*
*
RNA
Trichloroacetic acid (TCA) insoluble
+ [a-32P]rNTPs
TCA soluble
The separation and identification of the three eukaryotic RNA
polymerases by column chromatography.
A protein extract from the nuclei of cultured
frog cells was passed through a DEAE
Sephadex column. Adsorbed proteins were
eluted (black curve) with a solution of
constantly increasing NaCl concentration.
Fractions containing the eluted proteins
were assayed for the ability to transcribe
DNA (red curve) in the presence of the four
rNTPs.
The synthesis of RNA by each fraction in
the presence of 1 mg/ml of a-amanitin also
was measured (blue curve).
Three types of RNA polymerase in eukaryotic nuclei
RNAP
Location
RNA synthesized
a-amanitin
I
II
III
Nucleolus
Nucleus
Nucleus
Pre-rRNA, except 5S
Pre-mRNA, some snRNAs
Pre-tRNAs, 5S RNA,snRNAs
Insensitive
Sensitive to 1 mg/ml
Sensitive to 10 mg/ml
Pol I: single primary transcript, ~200 copies
Pol II: ~30,000 different genes
Pol III: 30-50 genes
(Also- Organelle RNAPs in Mitochondria, Chloroplasts. Encoded by organelle genomes.
Similar to bacterial RNAPs.)
Subunit structure of nuclear RNA polymerases
All three yeast polymerases have four core
subunits that exhibit some homology with the b,
b‘, and a subunits in E. coli RNA polymerase.
The largest subunit (L’) of RNA polymerase II also
contains an essential C-terminal domain (CTD). 26
(yeast) to 52 (human) copies of (YSPTSPS).
Phosphorylation site important for transcription.
RNA polymerases I and III contain the same two
nonidentical a -like subunits, whereas polymerase
II has two copies of a different a -like subunit.
All three polymerases share five other common
subunits (two copies of the largest of these). In
addition, each RNA polymerase contains four to
seven unique smaller subunits.
Structure of eukaryotic RNA polymerase II
Taq RNAP
rRNA promoter transcribed by pol I
UCE
core
45S pre-rRNA…
+1
-150
Pol I
-150
UBF
UBF
SL1
RNA pol III transcription:
the 5S RNA gene
TFIIIB: contains TBP
+ 2 other subunits
- all three RNA pols use TBP!
TFIIIA: Zn-finger protein,
Binds RNA and DNA
Mapping start sites
by in vitro
transcription
Fractionation of
accurate initiation
activity
Initiation
factors
- + + +
Deletion analysis to
locate transcriptioncontrol sequences
Yellow- 5’ promoter
Blue- Reporter gene
Plasmids with deletions of various
lengths 5’ to the transcription-start site
are then transfected into cultured cells
(or used to prepare transgenic
organisms) and expression of the
reporter gene is assayed.
The results of this hypothetical
example (bottom) indicate that the test
fragment contains two control
elements. The 5’ end of one lies
between deletions 2 and 3; the 5’ end
of the other lies between deletions 4
and 5.
Linker scanning mutations
Yellow- promoter
Blue- Reporter gene
Overlapping linker
scanning (LS) mutations
(crosshatch) are introduced
from one end of the region
being analyzed to the
other. These mutations
result from scrambling the
nucleotide sequence in a
short stretch of the DNA.
Comparison of nucleotide sequences upstream of the start site in 60
different vertebrate protein-coding genes.
Each sequence was aligned to maximize homology in the region from -35 to -20.
The tabulated numbers are the percentage frequency of each base at each position.
Maximum homology occurs over a six-base region, referred to as the TATA box,
whose consensus sequence is shown at the bottom. The initial base in mRNAs
encoded by genes containing a TATA box most frequently is an A. [See R.
Breathnach and P. Chambon, 1981, Ann. Rev. Biochem. 50:349; P. Bucher, 1990,J.
Mol. Biol. 212:563.]
Cis-acting control elements
(a) Genes of multicellular organisms contain both promoter-proximal elements
and enhancers as well as a TATA box or other promoter element.
(b) Most yeast genes contain only one regulatory region, called an upstream
activating sequence (UAS), and a TATA box, which is ≈90 base pairs upstream
from the start site.
Enhancers and promoter-proximal elements in eukaryotic pol
II genes
Binding sites for activators that control transcription of the mouse transthyretin (TTR)
promoter in hepatocytes. HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. Cell
Biol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. Acad. Sci. USA 86:4117.]
Transcriptional Regulation Requires Over 100 Proteins
That Work in Concert to Trigger Proper Gene Activation
Major Points
1. Three different RNA polymerases I,II and III direct synthesis
of rRNA, mRNA and tRNA respectively
2. Each RNA pol is a multisubunit protein complex that recognizes
distinct promoter DNA sequences
3. Primer extension is a sensitive, rapid and accurate assay to find
the 5’ start site of transcription in vivo and in vitro
4. Cell-free in vitro transcription assays (Run-off) provide a
powerful tool to study the regulation of RNA synthesis
5. Promoter mapping (linker scanning) and cell-based reporter
gene assays help define composite Cis-regulatory elements
6. Upstream enhancers, silencers, core promoters and downstream
elements make up complex, modular control DNA elements