Lecture 27 & 28

Question 1

Why are CpG islands present

Answer 1

• 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

Question 2

describe mammalian x-inactivation

Answer 2

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

Question 3

What is the XIC

Answer 3

Contains a gene for a long non-coding RNA with:
– Xist = X-inactive specific transcript
– Tsix = antisense version of Xist

Question 4

Live birth created parental conflict, how?

Answer 4

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

Question 5

How does CpG methylation and histone modification affects silencing

Answer 5

CpG Methylation initiates the silencing and Histone Modification maintains it

Question 6

Prader-willi syndrome vs Angelman syndrome

Answer 6

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

Question 7

mRNA processing in prokaryotes vs eukaryotes

Answer 7

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

Question 8

What do mRNA contain? What do they affect (3 things)

Answer 8

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

Question 9

T/F mRNA is inherently stable

Answer 9

false, ribonucleases in cells can act on the 5’ or 3’ end

Question 10

What does increasing RNA stability yield

Answer 10

Means one transcript can yield more than one round of translated protein

Question 11

is mRNA more stable in prokaryotes or eukaryotes

Answer 11

eukaryotes

Question 12

What is the 5’end cap of pre-mRNA? what are the three additions

Answer 12

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)

Question 13

Describe the variety oof cap complexity in organisms(bacteria, yeast, plants &animals, vertebrates)

Answer 13

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)

Question 14

What does the Cap-binding complex do

Answer 14

Once the 5’end is processed it binds the cap-binding complex, allowing association with many other proteins and pathways

Question 15

What happens to the RNA pol II when a pre-mRNA is cleaved

Answer 15

the pre-mRNA is cleaved while the RNA pol II is still continuing transcription

Question 16

How is termination detected in pol II

Answer 16

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

Question 17

Describe the cleavage of termination

Answer 17

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

Question 18

Describe poly-A tailing in termination

Answer 18

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

Question 19

Why are caps and tails needed

Answer 19

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

Question 20

how does the cap and tail stabilize the mRNA? how does it enhance translation?

Answer 20

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)

Question 21

How does cap and tail association affect translation

Answer 21

Accelerates translation

Once bound and through the circularization, the ribosome can reassociate at a faster rate and protien synthesis becomes more efficient

Question 22

how odes impairing cap/tail affect translation

Answer 22

Slows translation

Question 23

Describe the cap

Answer 23

Short synthesis (20-30nt)
Inverted GTP addition by Guanylyl transferase (capping enzyme)
methylation - 7mg, 2’OH-m

Question 24

Overview of the tailing process

Answer 24

Specify with sequence & Recruit cleavage factors
Cleave, Polyadenylate, Bind stabilizer

Question 25

Do complex organisms have more DNA?

Answer 25

In general yes, however there are protozoans that have more genes than all other species, and many other exceptions as well

Question 26

what are the 3 important sequence regions in splicing

Answer 26

– 5’ splice site (GU)
– 3’ splice site (AG)
– Branch site (A)

Question 27

Describe the splicing reaction

Answer 27

1. 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
2. The 3’-OH end of the 5’ exon is bound to the 5’-P end of the first base of the 3’ exon
3. The lariat structured intron is released and degraded