16 Flashcards
what helpsbind rna pol from to dna in bactera vs euk
a sigma factor- bc core enzyme with no sigma, it doesnt bind but holoenzyme binds
in euks its the tail that is attached to unit that acts as sigma factor cuz there is none in euk, no sigma factor, just a dephosphorylated rna pol 2
essentially in euks the phosphyrlation is what acts as a sigma factor
What does transcription intiation rate depend on:
1)Availability and strength of promoter
2)presence/availability of regulatory proteins
3)availability and strength of their binding sites on dna (ie how close to a tata box, consensus sequence)
What are northern blocks and what is the trouble with it
rna gels, trouble is it only tells u how much rna it has at that particular time
So imagine a cell makes rna slowly and degrades slowly, it would have a dertain amount , but if a cell was making and degrading fast, it would be the same amount, ie if there is 100 leaving and 100 coming in, we wont tell a difference in if its fast or slow since we only have a value on what is there we cant tell anything about rate of making and degredation
So to know how well and how fast a transcription is intiated,we need to see it in the middle of it
Three types of studies to see how transcription rate is
1) Run Off: cut plasmid at a point to linearize it…
-have a gene and promoter, we put it in plasmid, we cut it and the trranscript essentially runs off the transcript cuz P at end
2) Nuclear Run Off:
cell is fed with 32P nucleotide
-add transcription factors to make it go, it will only turn on the things we are interested in
3)Reportergenes:
we know a region is involved in starting transcripion, we dont know what sequences tho (where thepromoter is) so
Imagine u havea. transcript that is 100 base long, but dont know where the promoter is, so wjat u do is chop a little at a time to see which chop changes the rate of transcription (ie, the first one doesnt effecct, move it down now it does)
-we use reporter gene to see the genes, (subsitute the actual gene with the reporter gene) we dont look at what the promoter actually controls
Reporter gene: easily assayable, produces chemical or enzyme when transcribed
-to find out promoter gene location we look at the results and see where rate decreased, means that there will be promoter gene somewhere there
Types of reporter genes
Reporter genesallow us to see how much of it ismadesince it does somethi8ng to make a colpour…
-B-galactosidase (it is an enzyme that can hydrolyze x-gal into galactose and blue pigment)…mroe expression of it would mean more blue colour
-Luciferase: protein that fluresces
-GFP (Green flurescent protein): flurescent, monkey had this in embryo and made him green
-CAT: Transferase, acetalyes antibiotic chloramphenicol (CAM) ,has different properties
RNAP 2 promoters
Promoter sequence Requirements:
a)Located close to start point
b)required for initiation of transcription
RNAP uses class 2 promoters
Most Class 2 promoters have 2 basic components: keep in mind this is for eukaryotes
1) Core elements: (called core promoter)
-ie for prokaryotes ( -35 - 10 regions)
4 elements in ideal
a) TATA box; similar to bacteria one
-consensus: perfect tata box
-found 30 bp from transcription site,
role to position rnap so hat it always starts 30 nucelutidfes downstream
-position rna pol so that it starts at the intiator
b) INR: initiator; sequence that has start point oftranscription
-element that also has a consensus sequence
-causes precise site of starting cuz its starts from here (STARTSITE)
-backup for bdingng
c)DPE: downstream promoter element, binds to transcription factor TFIID
-binds to another part of
-backup for bidning
d) BRE: TF II Recognition: binds rnap pol
-found upstream of tata
-positioning
-in reality, rna pol may not have all four of these components
-these are what u need to bidn to promoter
2) Regulatory elements: (enhancer)
-in prok: operator regions
-increase or decrease the efficiency of binding, dont need it TO bind but it does something to the bidning
-enhancers: work over distances of 10s of thousands of bp
-insulator: keeps enhancers within that transcriptional unit (found near to enhancer, it is dna that is heterochromatin so it cant be openedds
-proxmal promoter eelemts: work close
Upstream elements: (promoter proximal elements)
a)GC BOX:
-100bp away from start (close to tata)
b) CCAAT box: repeats that are 50-75 upstream, enhancer
c) Promoter-Proximal
Enhancers:
-stimulate transcription typically
-help increase efficiency of transcription of promoters from 1000s of bpdown stream
-work inanydistance and anyorientation
-work by looping dna and helpoing rna pol bind to promoter
SIlencers:
enhancersthat decrease transcription
Insulator:
-hetrerochromatin dna tht is so dense that it cannot bind to any trancription factors, cause loops and condensedm so that means that other enhancers cant really effect genes on other side, these insulators are places between genes (look at pic on slide 21)
think grabbing a pole 30ft up, hands are core elements, someone pushing me or grabbing me closer to pole is regulatory.
Without core elements: wont get transcription because sore needed to bind
Without regulatory: will get trnascription, may get really good or really bad prescription, cant control it though
How many rnap pol are there
3 atkest
Rnap 2: makes mrna
1: and 3: make rrna and small rnas
IF tata box is present what occurs
regardless of sequences, pol will start transcribing 30 sequences away, this is why the tata is foud 30 upstream from region
-it positions rnapol in a way that intiiation begins at a fixed place
What is a negative enhancer
silencer
Can enhancers work cross-chromosoally
Yes, an enhancer of one cna work on another strand
All dna exist in every cell… how does a eye cell has something that turns on whereas liver which has the same genes does not have something that is turned on ie if u have gene A, it exists everywhere buthow come its only turned on in specific tissues
Think: Dna is the same in everycell so that mean that cis elements will be present in all of thesecells (enhancers)
-for a cis element to be turned on or off it needs to be binded to specific protiens
Tissue specificity is due to the specific variety of regulatory binding proteins…called Transcription Factors (TF)… by controlling the production of transcription factors , u control what is turned on in specifc cells
If something should be on in a majority of cells… most likely will always be on and int the cells where we dont want it we will have transcripton factors that bind to those spcific cis elements to turn it off
ex: Liver cells make albumin gene, eyes do not so in eyes there are tfs that bind to the albumin gene… same with crystallin except reveredbc eyes have it
Transcription is highly regulated in that…
1 gene could be transcribed in response to MULTIPLE conditions… have multiple cis elements then .. therefore gene A and Bare differentiated now even by the different cis elements
different condiitons associated with different cis elements which bind to different reg proteins
Different ocmbo of cis elements respond to different cues,
cis elements dont have to be placed in the same places, jus have to be present and on to have similar effects
Cells can change / control transcription based on
environemnt
cell type
development
if there are two differentgenes that respond to the same signal in the same way what does this mean
they all have the same cis elements turned on (doesnt have to be in the same position)
this means tahat even the presence or absense of one cis element can increase or decrease transcriptoon rates
are promoters modular
ues, they can mix and match diffrent elements some have tata some have initator etc
this means that promoters can be controlled for different environments bythepresence or lack of certain elements
By doing things to upstream elements (make things to bidn to turn it on or tirn them off) we can control
which cell to expressit
whn to express
under which conditions to express
Summary of rnap promoters
Many have a TATA box as a core element (majority of those that don’t - have initiator sequences or CG-islands instead)
Proximal elements: required for developmental, environmental, tissue-specific etc. expression
Promoters that respond to same conditions (but control expression of different genes) have same cis elements although they may not be located in the same position relative to the start site of transcription
Enhancer: sometimes composed of multiple binding sites for different regulatory proteins; the combination of trans factors leads to increased transcription initiation
Outcome: expression of one gene can be induced or increased or decreased or shut-off by different conditions due to the modularity of gene’s promoter
in order for rna pol 2 to bind to core promoter it needs
generaltranscription factors
What are the two types of transcription factors
1) General transcription factors: these plus rna pol allow for transcription to occur
-without this, we have no transcrriptionwhatsoever
2)regular transcription factors: bind to regulatory sequences to alter the frequency of things (how much it is turned on), thinkof a dimmer light swich, u can alter how bright it is
Imgine yoy have promoter with a tata box…
first thing when u want transcription
Tata box binds to tata binding protein, tata binding protein brings in 14 other tata binding protein associated factors (TAFS)
this whole thing is TFIID (t2td)
-kinks dna at tata site
brings BRE closer to tata box, now brings in TFIIB which bends dna even more
now
TFIIF binds with rnap pol 2 (with mediator on rnap pol 2)
-B helps this complex bind to promoter
Once this binds
-TFIIF brings in TFIIH and TFIIE
-TFIIE is atapase, helps rna pol mov, and gives it energy to unwind dna
-TFIIH (helicase and protein kinase activity) phosphorylates the tail so pol can bind on to dna
D helps ,B helps F
what is TF2D (TFIID)
Tata binding protein and 14 tata binding protein associated factors
-binds at tata box via tata binding protein, whole complex is tata binding protein and 14 tbp associated factors
-kinks dna at tata site
-once bound TFIIB can bind
What does binding of tf2d allow for
binding of tfIIB
because TFIID creates kink in dna
TFIIB
binds opposite of TFIID
-really bends dna into a right angle lookin thing
so tfiid bends it slightly, tfiib bends alot (HELPS FOR POL CUASE POL SHAPED LIKE THIS)
