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BL 426 Study guide test 2 2011 Chapts 9-11plus some techniques Chapt. 5
Eukaryotic gene expression; prokaryotic DNA binding protein HTH motifs
Note Chapt. 10 with promoters does briefly preview activators binding enhancers.
The test will be worth 100 pts and will have a number of multiple choice questions, plus some matching
probably, maybe gel or experiment to interpret or design, and some compare/contrast, or short essay type
questions; maybe also some abbreviations to define.
1. Focus on what was presented in PowerPoints, using your text for additional clarifications and support.
2. Read learning outcomes for each chapter; also read summary at end of each chapter for the topics
we covered – the emphasis was pol II.
3. Focus on understanding important Figures – what is being shown; what type of experiment was done.
4. Answer the suggested review and analytical questions at the end of chapters.
5. Make a list of important new terms, concepts, enzymes, DNA sites and what they do
(many are listed below, but the list may not be comprehensive)
6. Complexity: 3 types of polymerases transcribe different categories of genes and use different promoters
and general transcription factors. Lots of big protein complexes are involved: GTFs, polymerases.
7. Consider the different model systems that were used (yeast, mammals) and what they told us.
For example, why is yeast such a good model system – haploid, diploid, grows fast, etc.
Why were animal viruses good models of eukaryotic expression? Herpes and Adenovirus
Why were phage and phage repressors good systems for prokaryotic regulation?
8. Consider some of the types of experimental techniques and assays were used to obtain the
understanding of the molecular mechanisms, many of which we have mentioned before.
Protein-DNA binding on membrane filters; in vitro RNA synthesis; Xray crystallography
Purification of proteins and protein complexes (affinity tags, ion exchange chromatography)
Western blot, immunoblot; EMSA; linker scanning mutagenesis;
Primer extension to map start and amount of specific RNA synthesis;
DMS footprinting; DNase footprinting of proteins binding on DNA; S1 mapping of RNA start site;
Mutant proteins, mutated DNA binding sites, mutated promoter sites; altered specificity repressors;
Reporter gene constructs to measure promoter activity; run-off transcription
9. Some general comparisions of eukaryote expression to prokaryotes – what is similar, what must be
different? - proteins bind DNA, polymerases transcribe RNA, polymerases share general crab claw shape;
gene expression can be repressed and activated
Prokaryotes: Consider prokaryotic HTH major DNA binding motif – how do side chains of amino acids
recognize specific bp in major groove? (hydrogen bonds, electrostatic, hydrophobic). DNA binding
proteins often function as dimers or tetramers, binding to diverse DNA sites that can be palindromes of a
sequence. Allosteric binding of inducers to lac, corepressor to trp repressors
Many activator sites are located upstream of transcription start (UPE).
Consider some general themes for the eukaryotes:
Specificity of process comes from combinatorial aspects – multiple proteins binding multiple
enhancer sites; diverse GTF proteins needed at pol II promoters – not all are used for each promoter.
Different mechanisms for gene regulation: basal level vs. activation, repression.
How is polymerase recruited to promoters? (Kornberg article tested two models)
For regulated expression, transcription factors communicate through TAFs (Fig. 11.14) or through
coactivator complexes (much more in Ch. 12); transcription factors bind upstream of start (UPE, UAS)
Some model systems: E. coli gal, lac, ara operons, trp repressor/operator;
phage repressor-operators (lysogen is immune to superinfection by that same phage)
Xenopus laevis oocyte, SV40 early promoter (pol II),
adenovirus major late (AdML) promoter and Herpes simplex TK promoter (both for pol II),
Some new concepts: protein complexes, bending DNA (TBP), Looping DNA,
Different activators use different TAF11s or different coactivators
Processivity -amanitin sensitivity
Some DNA sites:
operator (phage, trp, lac)
TATA
Inr enhancer
BRE
upstream element (UPE) DPE enhancer
silencer
Promoters for each type of polymerase
GC boxes
CRE site (for CREB activator)
Some RNA sequences:
tRNA,
rRNA,
mRNA,
5S rRNA,
snRNA,
Some jargon/ abbreviations: His-tag
TAP-tag
HTH motif
RPc
RPo
RNAP
5’-UTR
3’-UTR
Upstream
downstream
RNA start is +1
NTP
DPE
PIC
HAT
CTD (heptad repeat)
Some protein complexes, what they do and what they contain:
GTF
TFIID
TFIIE
TFIIF
preinitiation complex
TAFIIs
TFIIS
Transcription factors (SP1, CTF, NTF-1)
hnRNA
dNTP
DABPolF complex
TFIIH
Some proteins and enzymes:
Protein phosphatase
protein kinase
protease
helicase
RNAP (core and holoenzyme) for pol I, pol II, pol III
pol II subunits, especially Rpb1 (and its CTD) Rpb4,7
orthologs of prokaryotic RNAP
Antibody
TFIIB
nuclease S1
DNase I
HAT (histone acetyl transferase)
SL1
UBF
TFIIIA
lambda repressor
P22 repressor
434 repressor trp repressor (aporepressor) lac repressor
Structural information on proteins, combined with mutant data, helps probe mechanisms
Consider modifications of proteins and what they do – as for polymerase (CTD)
Phosphyorylation
-amanitin binding to RNAP
Scientists and contributions/ model systems:
Mark Ptashne
Rick Young
Roger Kornberg
Bob Roeder