Term
Which part of RNA pol is the specificity factor? what are the other components of RNAP?
What is the function of each component? |
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Definition
Sigma factor.
Other things in RNAP holoenzyme:
Beta (Rifampin binding site)
Beta' (Chelates Mg++)
alpha2 (Binds UP element)
omega a
The sigma factor increases the binding affinity of the holoenzyme to the promoter region. It dissociates from the DNA after initiation of transcription in a stochastic manner. This dissociation (ideally) sets up a "sigma cycle", which allows for sigma to increase initiation of a second transcript before the first is done |
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Term
| What's the difference between the "closed" and "open" RNAP complex? |
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Definition
DNA is not melted (DS) and RNAP complex binds poorly in closed complex
the open complex requires sigma factor and DNA melting (RNAP complex binds ssDNA) |
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Term
1) Name the prokaryotic core promoter element(s) and consensus sequence(s)
2) Additional upstream elements? |
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Definition
1)
-10 and -35 box:
-35 = TTGACa
-10 = TAtAaT
more like consensus = more transcription; less=less (spacing also matters)
- UP element = -40 to -60 bp; Recognizes RNAP, so it's a promoter. These are seen in rRNA genes (that need to be transcribed a lot)
2)
- Fis element: associated with UP element; -60 to -150; this is an enhancer, not a promoter, because it doesn't directly bind RNAP but still enhances transcription |
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Term
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Definition
-40 to -60 bp upstream of translation initiation site. It binds directly to RNAP and enhances transcription by up to 30 fold
This region is contacted by the C-terminal domain of the alpha subunit of RNAP.
other elements are -10 (TAtAaT) and -35 elements (TTGACa), which are contacted by the sigma factor |
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Term
| How long does sigma factor stay associated ith RNAP? |
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Definition
~2-8 bp. This marks the "length of initiation". Abortive products are observed up to 9-10 bp. After this length, RNAP doesn't dissociate spontaneously because the complex has changed conformation and binds more tightly.
These abortive experiments were done by putting heparin in with RNAP/DNA (w/ lacZ promoter). The heparin will bind RNAP when it isn't bound to DNA (b.c. of negative charge) |
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Term
| What would heparin do in an experiment usng a DNA binding protein |
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Definition
| it would prevent non-specific binding between + charged protein and - charged DNA (b.c. heparin is - charged) |
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Term
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Definition
prevents bacterial initiation of transcription (but not elongation)
It binds the Beta subunit, which is the active site of RNA polymerization |
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Term
| what is hyperchromic shift? |
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Definition
| something that increases its absorbance in different situations. Most famous example is DNA, which has a hyperchromic shift at 260nm when the DNA strand is melted. This phenomenon was taken used to figure out how many bp were opened up by RNAP |
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Term
give mechanism for Rho-independent termination
Rho-dependent termination? |
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Definition
hairpin forms near a T-rich sequence --> RNA is "pulled out" of the RNAP
Rho is a helicase-like protein that can unwind DNA-RNA dimers. It binds RNAP and when a rho-utilization (rut) site appears, it binds and starts translocating along the RNA molecule. When RNAP encounters a termination signal, it stalls and Rho either pulls the RNA out or directly invades and unwinds the RNA-DNA dimer |
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Term
| what is the essential nature of a bacterial terminator? |
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Definition
1) Something that binding to the transcript that destabilizes the RNA-DNA hybrid (either hairpin or rho)
2) something that causes transcription to pause (string of U's) |
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Term
Name function of each:
1) LacZ
2) LacA
3) LacY
4) LacI |
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Definition
1) Beta-Galactosidase (lactose -> glucose + galactose; a small portion of allolactose is created)
2) Galactoside transacetylase (biological function unknown)
3) Galactoside permease (pumps lactose into cell)
4) repressor (binds operator in the absence of galactose) |
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Term
for lac operon, what is the:
- Repressor
- Operator |
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Definition
repressor is lacZ protein, binds to operator in a tetramer form. When lactose binds the repressor, it dissociates from the operator
There are actually 3 operators (only O1 is necessary) |
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Term
| what is the role of glucose in the lac operon? |
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Definition
when glucose is high, cAMP is low.
When glucose is low, cAMP is high -> binds CAP (catabolite activated protein) -> binds CAP region of lac operon and activates RNAP for transcription
The promoter of the lac operon is not very strong, CAP binding helps stabilize this complex
CAP binds to the c-term of the alpha subunit of RNAP (same thing that binds the UP element!) |
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Term
Which eukaryotic RNApol makes rRNAs (except 7)?
mRNA?
tRNA? |
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Definition
RNA pol1
RNA pol2 (also makes snRNA)
RNA pol3 (also makes 7S rRNA) |
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Term
| Name components of eukaryotic promoter region |
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Definition
- BRE (TFIIB recognition element--TFIIB is a transcription factor). Located -37 to -32
- TATA box. located -31 to -26
- Initiator. -2 to +4
- downstream core promoter element (DPE). +28 to +32
NOTE: Unlike prokaryotes, eukaryotic RNA Pols can't bind DNA promoter regions directly, they need transcription factors ("TF___"); There are general transcription factors (allow basal expression of all genes) and gene-specific transcription factors (increase expression of certain genes) |
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Term
| in eukaryotes, which transcription factor for RNA PolII binds first? where does it bind? |
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Definition
TFIID; binds TATA box. This is absolutely necessary to assemble RNA PolII
Remember: D -> DAB -> PolIIF (F binds to PolII, not DAB complex on TATA box) |
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Term
| which eukaryotic transcription factor contains TATA binding protein (TBP)? What other stuff does it have? |
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Definition
TFIID; it also has up to 14 TBP-associated factors (TAFIIs)
TBP binds to TATA box, but TFIID binds to a large area, from TATA box (TBP) to past the initiator site, and to the DPE (downstream promoter element,bound by TAF1 & TAF2)
Different TAFs assemble in TFIID to cause differential response to different promoters. For example TAF1 & 2 + TBP is a very good promoter for just a TATA + initiator stretch of DNA. Conversely, Sp1 needs TAF4 (maybe others) |
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Term
| what is Sp1 and what does it bind? |
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Definition
| Sp1 is a transcription factor that binds upstream GC boxes and helps assemble RNA Pol. Sp1 can activate translation independent of a TATA box. It binds at least one TAF (TAF4), which in turn binds TBP and the rest of TFIID |
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Term
What does TFIIA do?
TFIIB?
TFIIF?
TFIIH? |
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Definition
- TFIIA: Enhances binding of TBP; not needed in vitro, but essential in yeast;
- TFIIB: Binds RNA PolII and bent DNA
- TFIIF: Tightly binds RNA Pol; may contact DNA upstream of TATA box; start site selection
- TFIIH: phosphorylates CTD of RNA Pol; Unwinds DNA creating transcription bubble |
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Term
| What are the 2 states of RNA PolII and their function? |
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Definition
II-A (unphosphorylated). Form that binds the pre-initiation complex
II-O (phosphorylated on CTD serines): Form that does translation
NOTE: the phosphorylation is thought to break the tether between TBP (& others) and RNA-PolII |
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Term
| dynamic phosphorylation of RNA-PolII is probably responsible for what? |
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Definition
crosstalk between RNA-PolII and the sequence it's transcribing. For example, initially S5 is phosphorylated by TFIIH; after the pol has "locked in", S5 is mostly dephosphorylated and S2 is phosphorylated
The phosphorylations mostly talk with histone modifiers & capping enzymes & such |
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Term
| name 2 models for termination in eukaryotes |
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Definition
1) Poly-A signal causes RNA PolII conformational change and release
2) Cleavage at poly-A site causes rapid degradation of RNA and termination (torpedo model) |
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Term
what are transcriptional activators?
What are their 2 major domains?
What are coactivators? |
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Definition
- proteins that bind to enhancers (DNA) and increase transcription above a basal level allowed by just promoters/transcriptionfactors
2 major domains:
1) DNA binding: Zn, Homeodomains, bZIP/bHLH
2) Activation: Acidic, Glutamine-rich, Proline-rich; NOTE: activators usually dimerize in homo or heterodimers: this increases DNA binding affinity. Zn-fingers can form a chain that contact the major groove in a sequence-specific manner
Coactivators are proteins that bridge between activators and general transcription factors (e.g. TFIID) |
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Term
| Name the 3 DNA binding domains of activators and which groove they contact |
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Definition
Zn-containing domains; Contact major groove in a sequence-specific but non-predictable manner. (remember, there can be many in tandem); Zn fingers are one type (best known), GAL4 are also Zn-containing
Homeodomains (drosophila), helix 3 has a portion that contacts the minor groove. Helix1 and most of Helix 3 contact eh major groove
bZIP ("basic leucine zipper"): contact major groove |
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Term
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Definition
| Use an DNA oligo to a transcript you want to measure. After transcription has taken place, treat with S1 nuclease, which degrades ssDNA and ssRNA. The oligo protects part of the transcript, that can then be detected |
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Term
| what prevents enhancers from activating the wrong genes? |
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Definition
Insulators, they:
- Prevent silencer from spreading heterochromatin (recruit HAT, histone methylase, and other chromatin remodelers)
- Stop enhancers from contacting the wrong promoter
(see slide 19 of lecture 15) |
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Term
| what 2 things turn transcription factors (transcription activators) on and off? |
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Definition
1) Signal transduction pathways activate and transport them to the nucleus (e.g. ras-raf)
2) covalent modifications of TF when they are in the nucleus (e.g. phosphorylation, ubiquitination, sumoylation, methylation, acetlylation); ALWAYS ON LYSINE!
Basic rules:
- Acetylation activates
- Methylation and Sumoylation represses
- Ubiquitination targets for destruction |
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Term
| How do transcriptional activators, that are very far away from their target, get to the promoter? |
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Definition
They loop out DNA. This was shown by the catenated plasmids (aatR, attB recombination) with promoter on one and enhancer on the other still were activated like they were on same strand
this argues that enhancers work in TRANS (conversely, promoters work in CIS, since they have to assemble the RNA-Pol) |
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Term
| describe chromosome conformation capture assay |
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Definition
"3C":
- Digest DNA
- x-link with formaldehyde
- Ligate
- reverse x-link
- do q-pcr with primers from different chromosomes to detect x-links & map regions of interstrand contact |
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Term
| How could you figure out what genes regulate the expression levels of another? |
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Definition
| do an shRNA screen and look for differential expression; do a CHIP-seq with identified targets to figure out if they are actually binding to the enhancer/promoter. You can also do a chromosomal conformation capture to see if the part of the enhancer is binding to the promoter of the gene of interest |
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Term
| what is cohesin and what does it do? |
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Definition
| it links DNA together (wiki says to keep sister chromatin together); lecture says it links loops of DNA together for enhancer/promoter interactions |
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Term
| Describe the technique "Hi-C" and what it is used for |
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Definition
used to map distant genomic interactions:
- X-link interacting DNA
- Digest w/ restriction enzymes
- Biotin label ends
- Ligate
- Sequence |
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Term
| how does chromatin fold (observation is based on how DNA interactions are mapped within a chromosome) |
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Definition
| it folds in a non-random, fractal manner (power-law dependence) |
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Term
| Describe a way to determine how much replication is going on in a cell |
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Definition
use Bromo-UTP or BrdU to label RNA being transcribed.
From this you could do a quantitative measure: figure out how much RNA is being transcribed, average normal gene length, figure out how many RNApols must be active at once |
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Term
| alternative poly-A signals? |
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Definition
| most mammalian genes have alternative poly-A signals to generate gene diversity |
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Term
| 2 bp upstream of poly-A site in mammals |
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Definition
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Term
| What does S1 nuclease do? |
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Definition
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Term
| what is polyA signal of mammals? Yeast? Plants |
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Definition
| all are AAUAAA, but mammals are very restricted. Yeast don't have any discernable sequence, just needs to be AU-rich. Plants are more tolerant of AAUAAA substitutions |
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Term
| Name proteins involved in polyadenylation |
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Definition
- CPSF (cleavage and polyadenylation factor) binds AAUAAA
- CstF (cleavage and stimulation factor) binds G/U
- CFI and CFII (cleavage factors)
another polyadenylation protein is needed to add A-tail |
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Term
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Definition
18: protein used by group-1 introns to stabilize their structure
19: used to fold g1i |
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Term
| Group1 introns use what metal ion? |
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Definition
| use 2 Mg++ in active site; there is a third Mg with unknown function |
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Term
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Definition
cuts 5' leader sequence of tRNA:
it directly interacts w/ tRNA sequence (because it's a ribozyme) and interacts structurally |
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Term
What is the difference between Group1 and grou2 introns?
which one uses an RT? |
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Definition
They both are self splicing but have completely different structure. Group1 has CYT18 & 19 proteins that facilatate splicing. Group2 resemble spliceosome
Group 2 introns use an RT to re-insert back into the genome; Group 1 use the cell's DSB-repair machinery |
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Term
| what is unique about the Hepatitis D virus genome? |
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Definition
| it is a ribozyme: the long, linear, concatenated genome self cleaves into monomers. |
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Term
| C-type lectin II (Clec2) has something in the mRNA. What is it and why is it important? |
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Definition
| it is a ribo-switch like hammerhead RNase. It self cleaves to decrease expression levels (binds some unknown thing); This is an immune protein found in mice |
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Term
| where does polyA machinery bind? |
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Definition
to the CTD of PolII
remember polyA machinery needs the specific AAUAAA sequence to function properly |
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