Key for MBMB 451 A Section 2-Bartholomew Fall 2007 10-30-07
1. Describe the properties of the general transcription factors of RNA polymerase II, I and III. What is TBP and what general transcription factors have TBP as a component? What enzymatic activities do some of the transcription factors possess and how do they facilitate transcription? Describe TAFs and if they are involved in activated or basal transcription. [14 points]
Properties of general transcription factors
a. Recognition of promoter
b. Start site selection
Transcription factors of RNA polymerase I
a. SL1: contains TBP (TATA binding protein), binds to specific promoter region only in conjunction with UBF1.
b. UBF1: bind independently to promoter region
Transcription factors of RNA polymerase II
TFIID: binds to TATA box, contains TBP and TAFs, responsible for promoter binding. TBP alone can facilitate in the assembly of the transcription complex
TFIIA: may activate TBP by releasing repression caused by TAFII230, stabilizes TBP binding
TFIIB: involved in start site recognition
TFIIF: Helicase activity, maybe involved in DNA melting, and helps bring RNA pol transcription complex
TFIIE: along with TFIIH it is involved in promoter clearance
TFIIH: has ATPase, helicase and kinase activity (CTD phosphorylation) and involved in DNA damage repair
Transcription factors of RNA polymerase III
TFIIA: along with TFIIC assist in TFIIIB binding at promoter location of 5S rRNA gene
TFIIIB: positioning factor responsible for localizing RNA pol III correctly
TFIIIC: binds to promoter regions (box A and B)
TBP is TATA binding protein. SL1, TFIID and TFIIIB have TBP as a component.
The enzymatic activities of the general transcription factors are described above. Phosphorylation of CTD will help RNA pol to move from initiation to elongation phase.
TAFs are TBP associated factors. They control the activity of TBP and TFIID and are involved in activated transcription
2. What are the principles of Ion exchange chromatography and how does it work in protein purification? What is a linear versus a step gradient? What is the difference between a weak anion exchange resin and a strong anion exchange resin? [12 pts]
Ion exchange chromatography works on the principle of charge separation and relies on the attraction between opposite charged particles.
Protein molecules, consisting of amino acids, have ionizable groups and carry a net positive or negative charge depending on the pH of the solution. This is utilized for their separation from a mixture of compounds. There are two types of ion exchangers: cation with negatively charged groups that attract positively charged proteins and anion that attracts negatively charged protein. The elution is generally carried out by a gradient of salt or pH.
Linear gradient: The salt concentration is linearly increased, example 0 to 500mM NaCl
Step gradient: protein eluted with different concentration of salt; 100, 200, 500mM.
Strong anion exchange resins are fully ionized over a wide pH range (trimethylaminomethyl, thriethylaminoethyl etc.); whereas, weak anion exchange resins are fully ionized over a narrow pH range (aminoethyl, diethylaminoethyl etc.).
3. Explain discontinuous gel electrophoresis and how it is used to separate proteins by size. What is an SDS-micelle and what is its role in gel electrophoresis? [10pts]
Discontinuous gel electrophoresis has a stacking gel layered on top of a resolving gel that are at different pH (6.8 vs 8.8). In the stacking portion the proteins are pushed together in a tight bind by a large boundary front of charged glycine. At pH 8.8 the glycine is no longer charges and the proteins migrate according to theie overall charge and the pore size of the acrylamide gel. The system is called discontinuous because of different gels made with different buffer(0.125 M Tris-HCl pH 6.8 for stacking, 0.25 M Tris-HCl pH 8.8 for resolving gel). Resolving gels of different pore sizes (4% - 20% acrylamide) are used to separate proteins by their size.
Proteins samples treated with denaturing agents like B-mercaptoethanol or DTT disrupt disulfide bonds. SDS denatures proteins and forms a micelle around the protein. Approximately one SDS molecule bind per two amino acid residues. Movement of protein through the gel matrix is proportional to the size of the protein and is based on the size of the SDS micelle formed around the protein.
4. Explain how DNA recombination can occur …..
A similar process is initiated when a nick is detected in one of the strands
Reciprocal and patch recombination is obtained depending on resolution of the holiday junction. Nicking in the other strands release splice recombinants (reciprocal recombination) where as in the same strand release patch recombinants.
5.
a. RecBCD complex binds to ds DNA at double strand break
b. Then it moves towards left (has direction specificity with respect to chi sequence). While moving unwinds and degrades DNA (3’ to 5’ exonuclease)
c. At chi sequence it pauses, cleaves the top strand 4 to 6bp right of chi sequence.
d. Recognition of chi sequence cause RecD (exonuclease) to dissociate and get inactivated.
e. RecA bind to free 3’ end and use it for strand invasion, once it find a homologues duplex sequence. It also catalyses strand exchange.
f. Branch migration and resolution of holiday junction catalyzed by Ruv AB and Ruv C respectively.
Functional and structural properties of RecA protein
Binds cooperatively to DNA to form RecA-DNA filaments, will bind to ssDNA or dsDNA and forms a three-stranded structure. There are six RecA subunits per turn of filament. RecA hydrolyzes ATP during the transfer reaction of one strand to another strand in the dsDNA template. (see pages 964-965 in Lehninger)
6. Self splicing and difference between gr I and gr II intron
Self splicing: RNA itself can perform the splicing reaction in vitro, without requiring enzymatic activities provided by proteins.
Difference between Group I and Group II introns
Both group I and II introns are spliced by two successive transesterification reactions
First reaction in group I introns: 5’ exon intron junction is attacked by a free hydroxyl group provided by a guanosine or guanine nucleotide.
First reaction in group II introns : 5’ exon intron junction is attacked by a free hydroxyl group provided by an internal 2’-OH position.
For both of them 2nd reaction is the same where the free 3’-OH at the end of the released exon in turn attacks the 3’ intron-exon junction
Group I intron are more common than Group II introns
7. Describe the process of m-RNA splicing
m-RNA splicing: process of removal of intron from pre M-RNA to form mature m-RNA
spliceosome contains Sn RNPs U1, U2, U5, U4 and U6, each containing a number of proteins (usually less than 20)
SPLICING
1. U1 snRNP recognition of the 5’ splice site.
2. Early complex formation (commitment comple) containing U1 and the splicing factor U2F. U2F binds to pyrimidine tract downstream of splice site
3. U2 snRNP, have sequence complementarities to branch site, bound to branch site. This forms the A presplicing complex. It requires ATP hydrolysis and commits pre-mRNA to the splicing pathway
4. Ser-Arg rich SR proteins act as splicing factors and regulators
5. B1 complex form when a trimer containing U5 and U4/U6 joins the complex. This is spliceosome
6. B2 complex: Release of U1 from the complex brings the components just opposite to 5’ splice site
7. U5 change position from exon to intron
8. C1 complex: Catalytic reaction: Release of U4, requires ATP hydrolysis. U6 is free, binds with U2 and close to 5’ splice site. It contacts the conserved GU in 5’ splice site.
9. 5’ site cleaved and lariat formed
10. C2 complex: Second transestification reaction, 3’ site cleaved and exons get ligated. The mature m-RNA released.
Exons : Expected coding sequence
Intron: intervening non coding sequence or intragenic regions
Conserved features of intron: (1) 5’ splice site: GU (2) 3’ splice site : AG
Bonus question
SWI/SNF properties
1. A 1.5 MDa complex involved in ATP dependent chromatin remodeling
2. Repositions nucleosome on a DNA surface both cis and Trans
3. Evicts histone octamer
4. Evicts H2A.H2B dimer
5. Topological changes in close circular arrays
6. form cruciform DNA from inverted repeats
7. Accessibility assays – restriction enzyme cutting
Models
(1). Twist diffusion model: small twist propagated, breaking 1 histone DNA contact at a time, from one end to other, hence moving nucleosome very short distance, less disruptive
(2). Bulge propagation model: bulge is propagated from one end to other moving nucleosome ~ 50bp, accessible to restriction enzyme, more disruptive
Coactivators
In essence SWI/SNF is a coactivator that is recruited by certain transcription activators like Gal4, Gcn4, and steroid receptors. It facilitates transcription be opening chromatin and allowing the other transcription factors access to promoter DNA.