Gene Expression And RNA Processing Flashcards
What is transcription in E. coli
What are the special things about it
DNA to RNA
RNA is made 5’ to 3’ and uses the 3-5 stand of dna to transcribe
Is slower than dna replication because it transcribed 50nucletides/sec
DNA does 1000/sec in replication
Doesn’t need primer to start transcription because of the ppp and the start of the rna strand
Doesn’t need helicase to split apart the dna strands
What is needed for transcription to happen
NTP (not dntp)
RNA polymerase
DNA template
Divalent cation like mg2+ (in rna pol active site)
What are the subunits of RNA pol in E. coli
Two alpha
Beta
Beta prime
Omega
Sigma 70 (70kda)
What do the 2 alpha subunits do
The assemble the core rna pol enzyme and interact with regulatory factors
What does the beta and beta prime subunits do
Catalysis of transcription
Interacts with dna and rna
What does the omega subunit do
Restore the denature polymerase to its native form
It’s important for the structure of the enzyme
What does the sigma 70 subunit do
Takes part in promoter recognition
What subunits form the rna pol core enzymesm
What about its active site
Aplha x2
Beta
Beta prime
Omega
Beta and beta prime
What is a holoenzyme
The core enzyme and the sigma subunit
The active site of rna pol is
In between beta and beta prime
Where is transcription initiated
The promoter
Has the -10 (also called pribnow box) and -35 sequences upstream of the +1 start site of the coding strand
These sequences are 6 bp long
How is transcription initiated
First the sigma subunit of the RNA pol decreases rna pols affinity for dna
This lets the rna pol move quickly along the dna template strand to find the promoter
The sigma subunit then recognizes the promoter (-35 and -10)
What happens to the sigma subunit after it’s found the promoter and many rna nucleotides have been made?
The sigma subunit falls of the core of the holoenzyme
Does rna synthesis need a primer to start
No
How is elongation carried out in rna transcription
Where does the already made 5 prime rna go
After forming the first phosphodiester linkage, elongation happens
New ntps are added to the growing strand at the 3’ end
The 5’ already made rna is released though a channel in the enzyme
What is the transcription bubble
How long is it
The denatruated area of the dna that’s unwounds and rna is being made
17bp long
Is the reaction of pyrophosphate (PPi) to 2Pi exergonic or endergonic
Exergonic so the reaction of adding NTPs is driven forward
What types of termination are the in rna transcription
Intrinsic termination
Protien dependent termination
How does intrinsic termination of transcription happen
The rna product froms a hair pin because of complimentary pairs of G AND C that are repeation in the dna
This makes the rna pol pause
A sequence of a bunch of U is after this hairpin in the rna
These A to U bp bonds (rU-dA) are weak because there’s only 2 h bond between them
So the rna strand dissociates from the template dna strand and the RNA pol enzyme
What is protien dependent termination
This is when the rna pol need help in stopping where there isn’t a hairpin and UUUU repeat
A protien called rho which is a helicase (hexameric and hydrolyzes atp)
The rho protien pulls the rna strand away from the DNA through its centre.
What is different in transcription between prokaryotes and eukaryotes
Prokaryotes: transcription happens at the same time as translation (The ribosome binds right as the rna pol makes a new strand)
More simple control elements (less protiens used)
The termination signal is the GC hair pin
The mRNA transcript isnt processed and transported across a membrane
Eukaryotes: transcription happens in the nucleus, the rna transcript is processed, then sent to the cytoplasm
Translation happens in the cytoplasm
Termination signal is the Poly A tail
What are all of the promoter elements for rna pol
There are 6
They define the start site of transcription on the dna and recruit the rna pol
Cis acting elements
TATA box
Inr (initiator element)
DPE (downstream core promoter element)
Enhancer
CAAT box and GC box
What are cis acting elements
DNA sequences that regulate expression of a gene that’s on the same molecule of dna
What is the tata box and the Inr
Together they make up the promoter
TATA box is -25 bp upstream of the Inr
The Inr is the initiator element and it at the +1 site of the dna
What is the DPE
The downstream promoter element which is +30 bp down stream from the inr (+1)
It can also make up the promoter with Inr
What is the enhancer
Can be more than one kb upstream from the start site
What is the CAAT box and the GC box
They are involved in regulation and initiation of transcription
They’re involved in transcription of genes that that are constitutively (always) expressed
They’re at the -40 to-150 region upstream of the Inr
What is the pre initiation complex (PIC)
How does it start
This is formed when transcription factors bind cos acting element that recruit RNA pol 11
This complex is first started by the TATA binding protein (TBP) on TF11D recognizing the TATA box and binding to it
TF11D is a dynamic protien meaning it has a lobe (lobe A) which moves as the TBP is looking for the TATA box
How is the PIC formed
First the TBP on TF11D recognized and binds to the TATA box on the dna after moving through many diff confirmations
TF11A binds to TF11D and stabilizes the interactions with the promoter region
TBP engages the promoter by bending the DNA sequnce
TF11B binds and recruited RNA POL 11 , TF11F and E
The. The last step
What is the last step of the PIC forming
The TF11H helicase bind and unwinds the dna
It the phosphorylates the c terminal domain of RNA pol 11
This phosphorylation causes a transition from initiation to elongation
and recruitment of rna processing enzymes
Do prokaryotes need a helicase to unwind the dna For rna transcription
No, the rna pol does it itself
In what way does the PIC regulate transcription
How can transcription be more regulated
At low levels, It give low level of gene expression
More transcription factors that bind to other sites on the dna can stimulate higher or lower levels of transcription
The gene as expressed in a tissue specific manner
What is combinatorial control
When one transcription factor on the dna has little influence on its expression
So many other transcription factors are needed to make a complex that either stimulates or repressed transcription
How is mRNA processed in eukaryotes
A 5’ cap and a poly a tail is added to the rna strand
Through splicing where The non coding region (introns) are removed and the exons (coding) are joined together
Splicing can make many different combos of genes based on how the mRNA is spliced
How is the 5’ cap added to the 5’ end of men’s
The 5’ triphopsate of the rna has one phosphoryl group removed by phosphatase
Then the 5’ dihposphate end attacks the alpha phosphorous of GTP (guanalyl transferase does this)
The N7 of the guanine from GTP gets a methyl group on it, and the 2’oh of the divided nest the guanine also get methylated (by guanylmethyltransferase)
Why does mRNA have a 5’ cap
Do protect the 5’ end of rna from nucleases and enhance translation
How is the poly A tail processed
The CPSF (cleavage and polyadenylation specificity factor) recognizes the cleavage signal of the rna
Then an endonuclease cleaves the part of the rna with the signal
Then poly A polymerase adds a 250 bp adenylate (Adenines) to the cleaved 3’ end
What does the poly a tail help with
Stability and translation
How does splicing occur
The spliceosome which is a complex of rna / protien recognizes conserved (specific) sequences in the introns
These sequences are right next to the exons, so they are split right next to the exons
Then the spliceosome joins together the exons
Describe the process of splicing up to the lariats intermediate
The OH of the introns adenylate breaks the ester bond between the intron and the exon at the introns 5’ splice site
(1transwsterification reaction)
This forms a lariat intermediate where the two introns form a phosphodiester bond
The 3’ exon is still attached to the intro
What happens in splicing after the l lariat intermediate is formed
The released 5’ exon has a free 3’ oh group that attacks the ester bond of the second splice site
(2nd transesterification reaction)
This joins the two exons together and releases the intron in the lariat form goth a free 3’ oh
What is a transesterification reaction and how many does the spliceosome do
A reaction between and oh and and ester to make a new oh and ester
2 reactions
