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Transcription：RNA polymerase、Initiation、Clearance、elongation、termination、modification……
编辑于2022-06-09 22:45:43- Termination
- RNA poly…
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- modificati…
- Clearance
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Transcription
RNA polymerase
Prokaryotic
RNA pol
mRNA+ribosomal RNA+tRNA
holoenzyme
core enzyme
4 subunits:
α，α，
hold complex togeter
binds regulatory molecules
β，β‘
binds DNA template
binds NTP precursors
It can intitiate transcription but not accurate
σ
recognize first -35 orient pol correctly, and then recognize and open at -10 of the promoter
helix open at -10
unwind 12-14bp
pol cover about 75bp
make sure the accuracy of initiation
Eukaryotic
RNA pol I
most pre-rRNA: 18S, 5.8S, 28S
promotors are species specific
RNA pol II
pre mRNA, pri miRNA, snRNAs
10-12 subunits
550 kDa
kinase phosphorylates its ser and thr then activates it
With C-terminaus consensus sequences
YSPTSPS
RNA pol III
pre-tRNA, U6 snRNA, 5s rRNA
most their promotors locates internal of the gene...
house keeping
Initiation
Prokaryotic
promotor
-10: TATAAT
-35: TTGACA
Recognized by σ subunit of RNA pol
those 2 6bp sequeces common for most bacteria
indicate both site and direction of transcription
regulatory sequences
operater
down stream promotor
Repressor binding
inducer can deactivates inhibitor
operon is induced from repressed when inducer level is high
ex. Lac operon
some cases molecules activates inhibitor
operon is depressed from repressed when this molecule level is low
ex. Trp operon
Repressor block RNA pol
CAP binding site
up stream promotor
cAMP activated CAP binding to DNA
contributes to phenomenon of inducer exclusion in lac operon
cAMP-CAP contacts RNA pol and increase it binding with promoter
no primer
start most from ATP>P
Eukaryotic pol II transcriptional control region
pol II promotor
core promotors
transcripton-associated factors binding
initiator sequence
specific accurate starts
TATA box(most cases)
similar function of -10 sequence
recognize mostly by general transcription factors
General transcripton factors
specifically
TFIID
TBP subunit
binds TATA box
TAF subunit
regulates TBP binding to TATA box
TFIIB
binds BRE element
TFIIF
3 subunits
binds to TFIIB and RNA pol II
so it stabalize the TFIIB-BRE binding, and recruits RNA pol II
TFIIE
2 subunits
Recurits TFIIH
TFIIH
9 subunits
It binds the transcriptional initiation site DNA and unwinds it
Phosphorylats CTD of RNA pol II.
clearance of RNA pol II from promptor.
diagrams
position RNA polymerase at the transcription initiation site, and then release the polymerase to initiate transcription
like the σ subunit
it assembles transcription apparatus and recruits RNA polymerase II to promoter.
nessesary
do not increase the rate beyond the base level
form an intiation complex
TFIID
local regulatory sequences
Specific transcription factors
It increase transcription rate in certain cell types or in response to signal
i.e. tissue/time dependent manner
huge number/diversity
it has huge variaty of DNA-binding motifs
include distal activator proteins
DNA binding domain independent of activating domain(which interact with transcription apparatus)
they may freely swiched
but they do not bind to promotors but enhancers
include factors direct bind to specific site within core promotor, upstream of general transcription factors' binding site.
combine with intiation complex form a final transcription complex
pol II distal elements
Enhancer
binds by Distal activator proteins
when DNA folds it will approximate initiation complex
it interacts with the complex and increase the rate of transcription
sometimes observed in bacteria..
necessary for high levels of transcription
Silencer
feature
Independent of
position
far/distal
over comed by DNA loop formation
orientation
upstream/ dowmsteam
diagram
eukaryotic examples
yeast's regulation in galactose metabolism
mimics lac operon
GAL genes are genes involved in Yeast galactose metabolism
GAL1,2,7,10
phspho-galactose as a inducer
*1000 transcription rate when galactose present
galactose specific upstream activating sequence(UAS)
upstream of GAL1 and GAL10
binds galactose-specific transcription enhancing factor
binding sequence contain 4 repeats of 17bp palindrome
they are occupied by 4 activators(Gal 4p) which also covered by 4 suppressors act specifically on Gal 4p(Gal 80p)
the confomation of suppressors changes by inducer binding
The activators's activating domain exposes
activate transcription
activators also contain DNA binding domain
epigenetic
chromatin structures can reduce DNA accessibility of initiation factors and RNA pol II
epigenetic proteins
Clearance
RNA polymerase undergo conformational changes
to break the strong contact with promotor during initiation
elongation
Prokaryotic
σ subunit may leave or remain
transcription bubble
newly thysesized RNA form a 9 bases helix, and stabalize the 3'end for incoming NTP
it moves down at constant 50nt/sec
mRNA protruding from the bubble, DNA is rewound as it leaves the bubble
coupled with translation
as soon as 5'end of mRNA becomes available
Rate
50nt/s
Phosphorylation on RNA polII's tail domain accelerate transcription
termination
terminator
prokaryotic
simplest/well defined
A C-G rich sequence followed by A-T rich sequence
G/C self complementary
form a C-G rich containing hair pin to stop pol II movement
poly A-T bp
form a poly U weak interaction make mRNA dessociate from template DNA/ transcription bbullble
Eukaryotic
exist but not well defined
variaties of protein factors involved
ex. Rho-dependent termination
1. Rho binds to the mRNA at a “Rut” (Rho utilization) site and scans down the mRNA until it catches up and associate with the RNA polymerase, because RNA polymerase is stucked by hairpin.
2. The Rho protein complex then recognizes the mRNA C-G rich region and assists in termination
modification
only for eukaryotes
The capping and splicing occur during transcription(coupled), and RNA polII help recruits factors for modification
5'cap
time
Occurs before transcript is 20-30 nt long
Co-transcriptional capping
kinase phosphorylates CTD of RNA pol II
capping enzyme caps on phosphorylated RNA pol II
capping enzyme synthesize 5' cap on mRNA transcript
attachment
add methylated GTP(5'-7-methylguanosine/m7G) to 5' phosphate group
results methyl G cap
actually GMP
connected by 5'-to-5' bond
only one found in all nucleic acids
5' of primary transcripts usually A/G
further methylation
on cap0(the GMP guanosine nitrogen), cap1( on the first 5' nt of the primary transcript's 2' -OH), cap2(so does 2' -OH....)
by Methyltransferases
the enzyme acts with cofactor MAS: S-adenosyl Methionine
Function
5'cap Regulates mRNA turn over, and protects mRNA from 5'-3' degredation
5'cap directs pre-mRNA to processing and transport pathways
5'cap initiates most translation
at least great efficient
called 5'-end dependent mechanism
poly A tail
happened when transcription is almost terminus
5'---AAUAAA--(10-30nt)--CU---(30nt)--G/Urich---3'
first poly-A polymerase cleaves primary transcript at CU site which 15nt down stream of a convergent AAUAAA ,CU is futher 30nt up stream the AAUAAA is a G-U rich sequence
protein factors involved
1. CPSF
Endonuclease
2. CstF
bind to G/U-rich sequence
3. CFs
may recruits CPSF
4. PAP
Polyadenine Polymerase
Then poly-A polymerase add 100-200As to the end
2 stage reactions
An exonuclease digests the sequence between the terminus and cleavage
Function
polyA tail stabalize mRNA by protecting it from 3'-5' degredation
A sequence of A and U nucleotides near the 3' poly-A tail of a transcript promotes removal of the tail, which destabalizes the mRNA
1. by targets it for 3' to 5' RNA exonucleases
2. by stimulates the decapping enzymes that remove the 5' cap leading to degredation by 5' to 3' RNA exonuclease
necessary for path through nuclear pore
recognize by pore receptors
neccesary for efficient translation
poly A tail links to 5'Cap through protein factors
2PAPBA bind to polyA tail
elF4G binds to 2PAPBA
elF4G binds to elF4E which attched to 5'Cap
resulting circular mRNA
Allows ribosomes recycled quikly because termination site and reinitiation site are linked
Pre-mRNA Splicing
spliceosome
consist of snRNPs
small nuclear ribonucleo protein particles
complex composed of snRNAs and proteins
1 splicesome contains 150+ proteins and 5 snRNAs
5 major snRNPs exist and assemblied in specific order
snRNAs: U1, U2, U4, U5. U6
named by they rich in uracil
60-300nt
its assembly require pre mRNA
introns
noncoding sequences interupt the sequence of gene
24% of genome
intron-exon junctions
all introns begin with the same 2-base sequence and end with another 2-base sequence that tags them for removal
or 5' splice site & 3' splice site
branch point sequence
with a conserved branch point A nucleotide within the intron
exons
coding sequences/expressed sequences
1 to 1.5% of genome
process
1. snRNPs first recognize 5'end of intron and the branch point A by snRNA hybridization
U1 recognize 5' splice site
BBp and U2AF recognize branch point
Then its replaced by U2
2. snRNPs assoctiate with other factors to form spliceosome
by U4, U5, U6 assembly
3. splicesome cleaves 5'end of intron, and attach it to the 2'OH of branch point A
first transesterification
form a lariat
4. splicesome cleaves 3' end of intron
4. 3' end of the first exon displace 3'end of the intron and joint to the 5' end of second exon; lariat exises, spliceosome disassembles
second transesterification
alternative splicing
contributes to 15% of known human genetic disorder
may occor in 40% human genes
25000proteins----80000mRNAs
studied by proteomics
other definations
Transcription unit
region from promotor to terminator
streams
transcription start at +1
transcription start site
or the first base transcribed is called +1
Clearance/escape
The process of RNApol leaving the promotor
Transcription bubble
The region containing the RNApol, the DNA template, and the growing RNA transcript
operon
only in prokaryotic
the grouping of functional related genes that can be transcribed by one mRNA
most enzymes involved in one biochemical pathway
can be regulated together
Primary transcript
The RNA synthesized by RNApol II
mature mRNA
finally processed form that can be exported out of nucleus
A gene is colinear with its protein product
no introns
control of transcription initiation
positive control
control through increases the normal frequency of initiation
negative control
control through decreases the normal frequency of initiation
depend on effect of the regulatory protein
coupled
Refer to 2 precesses usually happened together(spacial/temporal)
like transcription and translation in prokaryotes
like capping and splicing in eukaryotes
coactivators
Coactivators & mediators
they bind distal activator and then bind to initiation complex
specifically mediate some transcription factors action
i.e. connectors between some of specific and general transcription factors
may eptgenetic proteins
some coactivators are histone acetylase
some corepressors are histone deacetylase
smaller in number
one coactivator can target multiple transcription factors