Lecture 27 & 28 Flashcards
Why are CpG islands present
- CpG Islands are present because: – Silenced DNA is methylated –>5-me-C mutates over time from C:G to T:A –> CpG’s are
lost over evolutionary time – Promoter regions are unmethylated, CpG’s are not lost & are kept under selective pressure as a mechanism for gene control - Totipotent cells start with low methylation at CpG islands – As tissues develop, unnecessary genes are methylated at CpG sites
describe mammalian x-inactivation
Contain a region called the X-inactivation center (XIC)
In the early embryo all x-chromosomes express Xist at low levels Each cell will randomly activate one X-chromosome at the Tsix locus
Tsix transcription prevents Xist transcription
If Xist isn’t downregulated, the RNA will coat the X-chromosome for inactivation
What is the XIC
Contains a gene for a long non-coding RNA with:
– Xist = X-inactive specific transcript
– Tsix = antisense version of Xist
Live birth created parental conflict, how?
Mom’s genome wants the baby to be just big enough to survive birth while the father’s wants the baby to be as big as possible creating a parental conflict
How does CpG methylation and histone modification affects silencing
CpG Methylation initiates the silencing and Histone Modification maintains it
Prader-willi syndrome vs Angelman syndrome
A deletion in the 15q11-q13 removes a region that is dual imprinted
Prader-Willi
When the paternal copy is is deleted results in Prader-Willi.
Uplifted flexed arms when walking
Mouthing behaviours
happy disposition
inappropriate laughter
wide-boxed gait
Angelman
When maternal copy is deleted it results in Angelman syndrome.
Constant drive to eat
High risk of mental illness
hypogonadism
mRNA processing in prokaryotes vs eukaryotes
In prokaryotes mRNA is translated as it is being transcribed, as such it is ready to read immediately
In eukaryotes RNA synthesized is not a mature transcript and is transcribed as a pre-mRNA, and other modification is needed
What do mRNA contain? What do they affect (3 things)
5’ cap and 5’ UTR to affect stability and translational efficiency
– Start codon and open reading frame terminating at a stop codon
– 3’ UTR and poly-A tail to affect stability and translational efficiency
T/F mRNA is inherently stable
false, ribonucleases in cells can act on the 5’ or 3’ end
What does increasing RNA stability yield
Means one transcript can yield more than one round of translated protein
is mRNA more stable in prokaryotes or eukaryotes
eukaryotes
What is the 5’end cap of pre-mRNA? what are the three additions
improves the stability of mRNA against 5’ exonucleases
first addition: capping enzyme associates with the RNA pol II CTD when Ser5 is phosphorylated, 5’to5’ bond is made between GTP and the growing mRNA
Second addition: the inverted-guanine is methylated at position 7 by guanine-7-methyl transferase
Third addition: methyl transferase can methylate the 2’-OH of the first two 5’ end nucleotides (only in multicellular organisms)
Describe the variety oof cap complexity in organisms(bacteria, yeast, plants &animals, vertebrates)
Bacteria: no cap
Yeast: only 7-m-G cap
most plants and animals also methylate the 2’ carbon of deoxyribose of the +1 nucleotide (cap-1)
Vertebrates: also methylate the 2’ carbon of the +2 nucleotide (cap-2)
What does the Cap-binding complex do
Once the 5’end is processed it binds the cap-binding complex, allowing association with many other proteins and pathways
What happens to the RNA pol II when a pre-mRNA is cleaved
the pre-mRNA is cleaved while the RNA pol II is still continuing transcription
How is termination detected in pol II
the pol II CTD has proteins for termination facilitated by a lack of Ser-5-P and Ser-2-P
CstF and CPSF
Once RNA pol II transcribes a signal in the pre-mRNA the factors will move from the pol II to the pre mRNA
AAUAAA is bound by CPSF
High G/U region is bound bt CstF
Which bind 4 more factors
CF1&2 which cleave the pre-mRNA
PAP which adds a few A’s to the 3’end
PAB bind the short run of A’s to stimulate further polyadenlyation
Describe the cleavage of termination
after CstF and CPSF bind four more factors bind
CF I & II
PAP
PAB
Cleavage factors I and II cut the pre-mRNA between the two boxed sequences
Describe poly-A tailing in termination
After the mRNA is cleaved, PAP adds a few A’s to the 3’ end
PAB binds this short run of A’s to stimulate further polyadenylation
PAP is enhanced when both CPSF and PABP are present and quickly add up to 250 A’s
Why are caps and tails needed
More protein can be translated from a mRNA when it is properly modified
modification stabilizes and regulated nuclear export, it also associates more strongly with ribosomes
how does the cap and tail stabilize the mRNA? how does it enhance translation?
once exported out of the nucleus for translation the cap and tail stabilize the mRNA and enhance translation by forming a closed-loop
5’ cap is bound by eukaryotic initiation factors (eIF) which in turn associate with the 3’ poly-A tail through PABP (here initiation refers to initiation of translation)
How does cap and tail association affect translation
Accelerates translation
Once bound and through the circularization, the ribosome can reassociate at a faster rate and protien synthesis becomes more efficient
how odes impairing cap/tail affect translation
Slows translation
Describe the cap
Short synthesis (20-30nt)
Inverted GTP addition by Guanylyl transferase (capping enzyme)
methylation - 7mg, 2’OH-m
Overview of the tailing process
Specify with sequence & Recruit cleavage factors
Cleave, Polyadenylate, Bind stabilizer
Do complex organisms have more DNA?
In general yes, however there are protozoans that have more genes than all other species, and many other exceptions as well
what are the 3 important sequence regions in splicing
– 5’ splice site (GU)
– 3’ splice site (AG)
– Branch site (A)
Describe the splicing reaction
- The G at the 5’ end of the intron is released from the exon and bound to the internal A “branch site” at the 2’-OH
end - The 3’-OH end of the 5’ exon is bound to the 5’-P end of the first base of the 3’ exon
- The lariat structured intron is released and degraded
