| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Readings: Brown, 8:179-181; 8:182-184
Outline:
A. Basic Features:
1. Eukaryotic RNA Polymerases
... [Brown, Table 8.1]
Three RNA Polymerases:
differ in cell location, RNA type made, & drug sensitivity
RNAPol I: rRNA
RNAPolII: mRNA
RNAPolIII: tRNA, 5S rRNA, snRNAs
Purification using a DEAE-Sephadex column
Note 1: Two assays used
First: assay for total
protein ... note that most of protein comes out in the "void
volume", i.e. does not stick to the column. This gives a
good protein purification step, since the RNA polymerase activities
stick to the column.
Second: assay for RNA polymerase activity,
with or without amanitin treatment
Assay: in vitro RNA synthesis using total DNA, e.g. yeast, as
template, 4 rNTPs, etc
This assay measures where RNA polymerase activity elutes from
the column as NaCl concentration is increased ... note the 3 peaks,
one for each eukaryotic RNA polymerase ... note the relative sizes
of the peaks ...
In general, three types of protein factors control eukaryotic RNA Pol activities:
A. Basal factors: Proteins that make up the CoreEnzyme
... 8-14 subunits ...
Cognates to prokaryotic subunits: alpha, beta, beta-prime ...
C-terminal domain on PolII beta-prime subunit ...
B. Transcription Factors, which are of two types:
1. Upstream factors: bind upstream sites to increase efficiency
of initiation ...
regulation of WHICH promoters are used, but no Temporal or Tissue
specificity
The RNA polymerase resulting from the Core Enzyme plus the TF Upstream Transcription Factors is usually called the Basal Apparatus ... or HoloEnzyme, for comparison to prokaryotic RNA polymerases ...
2. Inducible factors: bind upstream and downstream sites; Time & Tissue specificity
These Inducible Factors have no cognates in prokaryotic RNA polymerases ...
2. DNA Gene Expression (Transcription) Sites: ... [Brown, Fig 8.9]
Sites: taken together they constitute the Promoter
Core Promoter: sites near start nucleotide required for initiation of transcription
Initiator (Inr): Y2 CAY5 .. the start nuc (often)
(Recall: Y is any pYrimidine, R is any puRine ...)
TATA box: 15-25 nucs upstream ... conserved sequence with TATA
Promoter-proximal Sites: usually binding sites of Upstream Factors that convert a core RNA polymerase into a HoloRNApolymerase.
Spacing between, and distance of, these sites can often be varied ...
Enhancers: sites near or distant, upstream or
downstream, that enhance initiation
Sites interact by folding of DNA to bring sites into juxtaposition
spatially
Analysis of Enhancer region using Deletion Derivative mutagenesis ... [J. Art. 5]
General pattern of eukaryotic gene expression sites: ... [Brown, Fig 8.17]
3. Regulation:
Regulation in prokaryotes is primarily Negative (Repressors)
and a given Repressor usually coordinately regulates several contiguous
genes (Operon), eg lac and trp operons
Sometimes a given Repressor
will coordinately regulate MANY OPERONS; such is termed a Regulon.
Examples are the genes and operons expressed in the SOS or
the Heat Shock response
In eukaryotes regulation is primarily Positive (Transcription Factors are mainly Activators) and such usually controls expression of a single gene for a given transcription event.
Upstream factors and Inducible factors are both involved in regulation of eukaryotic transcription as Transcription Activators
4. Transcription Process:
As usual: Initiation, Elongation, Termination
Initiation: specific
for each of the three RNA polymerases ... see below ...
HoloRNApolymerase is "built" at Promoter for each transcription
event ...
Elongation: Proximal Protein Factors leave the Core RNApol, which transcribes at a slow rate using rNTPs as precursors to a transcription terminator site.
Termination: occurs by little-understood mechanism
at transcription termination sites downstream of Poly(A) binding
site, at which processing of the 3'-end of the initial RNA
transcripts takes place.
B. Methodologies:
Precise mapping of initiation start-site via "run-off" transcription in vitro
Precise mapping of initiation start-site via primer extension and S1 nuclease or S1 protection assays
Construction of 5'- and/or 3'- deletion mutants in regulatory region: ... [J. Art. 4 - Karin et al]
McKnight et al "Linker Scanner" mutagenesis
General procedure for identifying regulatory elements (binding sites) and isolating protein factors that bind to these sites: ... [Brown, Fig 7.16]
C. RNA Polymerase I:
... [Brown, Fig
8.9]
only transcribes rRNA; found
in Nucleolus; alpha-amanitin insensitive; 50-70% total RNA
Transcription units are clusters of rRNA genes; transcription
rate is very high
1. rRNA genes:
transcription of 18S, 5.8S, 28S rRNA species as pre-rRNA transcript
that then is processed into the three rRNA species: ... [Brown,
fig 9.20]
Sizes of these genes and the pre-rRNA transcript vary from species to species
These rRNA genes are found in Clusters of Tandem Arrays in the Nucleolar DNA in the Nucleolus part of the cell Nucleus
2. Initiation: ... [Brown, Box 8.5, Fig 8.9]
Two sites:
Core promoter: -45 to +20 (+1 is Start Nuc) ... G,C-rich
85% seq identity ... NO TATA box
Upstream Control Element (UCE): -180 to -107 ... G,C-rich
Two Protein Factors: UBF1
(upstream binding factor) and SL1 (Sigma-Like factor)
UBF1: single polypep; one copy binds each UBF1 sites; THEN
SL1 binds to both
SL1: 4 polypeps, including TBP and TAFs (TBP
Activation Factors);
species & promoter specificity
Product is a 45S RNA that is processed to give the 18S, 5.8S, and 28S mature rRNAs
D. RNA Polymerase III:
... [Brown, Fig
8.9]
5S rRNA; tRNA; snRNA; Nucleoplasm; species-specific alpha-amanitin sensitive
1. Initiation
Promoters: Downstream for 5S rRNA and tRNA genes; Upstream for snRNA
a. Type 1 promoter: 5S rRNA ... [Brown, Fig 8.9]
Box A +55 from Start Nuc, indep of nucs 1 to +54
Box C at +80 to +90 ..
NO TATA box ...
TFIIIA binds BoxA-BoxC region, promotes binding of TFIIIC
to same region ...
the two promote binding of TFIIIB to start region.
TFIIIB: ~ 5 proteins, including TBP (TATA Binding
Protein),
specificity for RNA Pol III which binds TFIIIB ... "positioning
protein"
thus: TFIIIB is the only true Initiation Protein, ala Sigma
b. Type 2 promoter: tRNA
Box A at +10 to +20; Box B at +50 to +65 ... within
the tRNA gene
NO TATA box
TFIIIC binds both Box A and Box B, but
binds BoxB with higher affinity than BoxA
... again promotes binding of TFIIIB to start
c. Type 3 promoter: snRNA genes
Promoter Upstream, consists of short sequences; variation among
genes & species
OCT - OCTamer binding site; PSE - Proximal Sequence
Element, near TATA box
OCT binds Octamer Binding Factor (OTF-1)
Stimulatory sites for RNA PolIII ...
TATA box is reqd ...
TBP binds TATA, ie TFIIIB, which positions RNA PolIII
Thus: TFIIIA,B,C form "preinitiation complexes",
reqd for positioning of RNA PolIII
E. RNA Polymerase II:
... [Brown, Fig 8.9, Fig 8.12; Table 8.3]
RNA Pol II: mRNA made; found in Nucleoplasm; alpha-amanitin
sensitive
12 subunits; 2 like prokaryotic beta and beta-prime;
Beta-prime subunit has a Carboxyl Term Domain (CTD) of
repeats ...
1. Basal Transcription Apparatus
(HoloRNApolII):
RNAPolII+General factors (GTF or TFII factors) ...
Minimal Promoter required: "generic" promoter ... get
tissue-indep expression
Again, short, separated sequences, mainly Upstream, the key ...
TATA always
2. Proximal Binding Sites:
Initiator (Inr): Y2 CAY5 .. the
start nuc (often)
TATA box: 15-25 nucs upstream
3. Basal Apparatus Proteins:
ordered assoc of factors
...
binds TATA in DNA MINOR groove
Similar to TFIIB for PolIII and SL1 for PolI ...
different TAFs may (probably) exist for different PolII promoters
TBP: 30 kD small protein ... structure known: saddle ...
TFIIA binds, extends Upstream ... may activate TBP ...
2-3 subunits
TFIIB binds, extends Downstream to ~+10 ... elongated protein
TFIIF binds PolII ... 2 subunits: helicase activity;
sigma homologue
probably brings PolII to Preinitiation Complex, interacting with
TFIIB
TFIIE binds, followed by TFIIH and TFIIJ
TFIIH: kinase activity, phosphorylates PolII CTD tail
... also has helicase activity ...
thereby releases PolII for elongation ...
Note that TFIIH is part of the eukaryotic nucleotide excision Excinuclease found in DNA repair, and hence is likely to be involved in Transcription-Coupled DNA repair ... [J. Art. 3]
TBP (TATA Binding Protein):
Saddle structure, that bends and distorts the DNA in the TATA
box region
TATA-less Promoters: more like Downstream PolIII promoters ... Inr important
Protein-protein Interactions
as important as DNA-Protein
Interactions
TBP the only protein to have DNA Sequence-Dependent interactions
Preinitiation Complex of proteins needed to position RNAPolII,
unlike bacteria
Many additional protein factors, specific to particular promoters
and tissues, which bind to sites in Enhancer regions, are
needed to yield full transcription efficiency in the cell (in
vivo)
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug
Smith