LEC24: An Example of Regulated Txn & mRNA Processing I

Question 1

what is normal hemaglobin structure?

Answer 1

2 alpha, 2 beta chains

Question 2

fetal vs adult globin chains?

Answer 2

fetus: gamma chain
birth: switch to beta chain

Question 3

where are alpha and beta globin genes?

Answer 3

1 beta gene on chromosome 11

2 alpha genes on chromsome 16

Question 4

what turns on beta globin expression? where is it?

Answer 4

KLF1, txn factor

binding sites close to beta clobin gene (-90) and in the LCR, locus control region

Question 5

when is KLF off/on?

Answer 5

KLF off in progenitor and proerthroblast; turned on in erythroblast stage; get beta globin gene expression once KLF1 synthesis turned on

Question 6

how does beta globin gene expression get turned on?

Answer 6

1) KLF1 gene on chromosome 19 turned on in erythropoiesis program at progenitor cell stage
2) KLF1 modified post-translationally by Phosphorylation at T41

activates KLF1

3) KLF1 interacts w/ CBP, a large complex including a protein w/ HAT activity
4) this increased acetylation opens up chromatin in vicinity; get txn of B globin gene

Question 7

what is structure of fetal hemoglobin?

when does this change?

Answer 7

2 alpha & 2 gamma chains

2 alpha globin genes on chromosome 16; 2 gamma globin genes on chromosome 11, pustream of B globin gene

several weeks before birth, level of gamma globin chains begins to decrease; elvel of beta globin chains increases

trend continues until few months after birth, when beta gene expression is max, gamma is low

Question 8

what is the adult form of hemoglobin?

Answer 8

HbA, alpha-2-beta-2

HbF constitutes <1% of HbA

Question 9

what turns on BCL11A?

what is BCL11A?

Answer 9

KLF1 turns on BCL11A; recall, KLF1 turns on beta globin

BCL11A is a txn repressor that turns off the gamma globin gene

SO same txn factors turns on beta and represses gamma

Question 10

what does KLF1 do?

Answer 10

activates B-globin txn and inactivates gamma-globin expression

Question 11

what do HbS sickle cell disease patients express re: globin chains?

Answer 11

normal Beta chains replaced w/ Beta^S

major hemoglobin form = HbS, a₂B^S₂

Question 12

what is hereditary persistance of fetal hemoglobin (HPFH)

Answer 12

condition in which gamma chains are expressed at significant level into adulthood

limits tendency of HbS to polyermize

pts w/ >15% of HbF show few disease signs; also beneficial in pts w/ B-thalassemia, who have poor B globin expression

Question 13

for whom is HPFH beneficial?

Answer 13

patients w/ B-thalassemia, who have poor expression of B globin

Question 14

what is the mutation associated w/ HPFH, hereditary persistence of fetal hemoglobin?

Answer 14

BCL11A enhancer mutation

intron 2 mutation!

less BCL11A expression –> more gamma expression

Question 15

what are the steps of pre-mRNA processing?

Answer 15

1) 5’ capping
2) splicing
3) 3’ Poly A

Question 16

what is the 5’CAP made of?

what is the bond it makes?

what does it enable?

Answer 16

CAP = 7-methyl-guanisine

linked to 5’ end of transcript in 5’ to 5’ phosphodiester bond

needed for translation initiation by RNA Pol II

Question 17

what is the 5’ CAP function?

Answer 17

only mRNAs

1) recognition site for binding of proteins that recruit a ribosome
2) protects pre-mRNA and mRNA from degradation by exonucleases that chew in 5’ to 3’ direction

Question 18

what does intron begin and end w/ usually?

Answer 18

begin: GU
end: AG

Question 19

what is the branch point of an intron?

Answer 19

where a 2’ to 5’ phosphodiester bond is formed when intron is spliced out

site of the 1st transesterification rxn

Question 20

what does spliceosome do?

Answer 20

huge complex of 200 proteins

has class of noncoding RNAs, SnRnPs - small nuclear ribonucleoprotein particles

catalyzes accurate splicing

Question 21

what is proces of splicing?

Answer 21

1) 2’OH of branch point A attachs the G of the loose consensus sequence at the 5’ splice site
2) 3’ OH of the upstream exon is now epxposed at 5’ splice site; it attacks phosphate of the phosphodiester bond, btwn end of intron and beginning of downstream exon, at 3’ splice site

**lariat RNA **forms during process of splicing

Question 22

what would happen if splicing is incorrect by 1 ntd?

Answer 22

frameshift mutation downstream

Question 23

where is the poly A signal?

what does it signal for?

what binds it?

Question 24

where is poly A signal?

what does it do?

how is it added on?

Answer 24

at end of 3’ UTR sequence

signals for endonucleolytic cleavage 10-30 ntds downstream

free 3’ hydroxyl is acted on by Poly-A polymerase, which adds 100-200 A residues in a template-indepenent fashion

Question 25

what is poly(A) binding protein?

Answer 25

binds poly(A) tail