Transcription and RNA Processing

Question 1

Summarize transcriptional activation in eukaryotes

Answer 1

TFs bind to the upstream DNA promoter activating sequences (UAS) and enhancer sequences, then attract HATs.

HMG proteins help it bend the chromatin structure so that TFs can interact with the Mediator, thus bridging between the transcriptional activators and the preinitiation complex (PIC)

Question 2

TBP in transcriptional activation

Answer 2

Only binds one orientation on the TATA box of the promoter, so it orients the entire PIC ; specifies the correct strand for transcription ; and defines the transcriptional start site (+1)

Question 3

TFIIE in transcription activation

Answer 3

ATPase and helicase activity (unwinding powered by ATP)

Also recruits TFIIH

Question 4

TFIIH in transcriptional activation

Answer 4

• Helicase - unwinds DNA at promoter
• Kinase - phosphorylates Pol II CTD to activate PIC
• Recruits NER proteins
• Initiate PIC assembly in TATA-less promoters (initiator elements/DPE)

Question 5

How is RNA polymerase activated over the transcription start site?

Answer 5

TATA binding protein (TBP) binds DNA in an orientation that assembles the PIC over the start site.

Specifically uses the template strand 3’ to 5’, so that transcription occurs 5’ to 3’.

Question 6

Template/nontemplate, coding/noncoding, sense/antisense

Answer 6

Template = noncoding = antisense is used to synthesize the RNA

Nontemplate= coding = sense is identical to the RNA (substituting U for T)

Question 7

What modifications occur to pre-mRNA at the 5’ end?

Answer 7

The 5’ triphosphate end of mRNA is methylated by cap synthesizing complex on the RNA Poly II CTD to form a 5’cap

The methyls groups are donated by SAM, whose regeneration requires folate and vitamin B12.

Question 8

What’s the purpose of the 5’cap?

Answer 8

Stabilizes the mRNA

Helps promote translation

Question 9

What modifications occur to premRNA at the 3’ end?

Answer 9

Addition of the poly A tail, as dictated by the poly A signal sequence upstream of the transcriptional termination

Question 10

What’s the purpose of the 3’ poly A tail?

Answer 10

Stability

Facilitates nuclear export of the mRNa

Augments translation in the cytosol

Question 11

What is the role of RNA Pol II CTD in pre-mRNA processing?

Answer 11

5’ capping is catalyzed by complexes associated with RNA pol II CTD, so that it occurs simultaneously with transcription.

Similarly, the endonuclease that cleaves the leftover transcript past the poly A tail is associated with the RNA pol II CTD

Question 12

Splice donor site

Answer 12

at the start of the intron

Question 13

splice acceptor site

Answer 13

at the end of the intron

Question 14

What is the role of spliceosomes and snRNPs in RNA splicing?

Answer 14

The spliceosome compelx contains snRNPs that create a lariat structure that removes the intron, and joins the exons.

Question 15

How do poisonous mushrooms affect transcription?

Answer 15

a-amanitin binds to RNA poly II’s back side and restricts its flexibility such that it can’t function –> significant reduction in RNA poly II transcripts and total protein

Liver damage, kidney failure, coma, death

Question 16

How does HIV ensure full length RNA transcripts?

Answer 16

1. Viral-encoded Tat protein acts as an anti-termination factor so that RNA Poly II keeps transcribing a full length viral transcript, which forms a 5’ hairpin loop.
2. This loop binds viral Tat & host cyclin T and CDK9 proteins.
3. Cyclin T activates CDK9 to hyperphosphorylate the CTD of RNA Poly II so prevent premature termination of the HIV transcript.

Question 17

What is B thalassemia? How do you recognize it?

Answer 17

Mutation at the B-globin locus causes less B-globin and more y-globin to be transcribed and synthesized.

Decrease in hemoglobin A (a2B2)and increase in hemoglobin A2 (a2y2)

Question 18

What’s the difference between B-thalassemia major (Cooley’s anemia), B thalassemia intermedia, and B+-thalassemia minor ?

Answer 18

Major : complete absence of B-globin expression; 2 alleles of Bo (Bo/Bo)

Intermedia: (B+/B+) or (Bo/B+)

Minor: reduced B-globin expression; least severe; (B+/B) or (Bo/B)

Bo = b globin levels are essentially zero

B+ = b lglobin levels are decreased

B = normal

Question 19

How is RNA transcription and splicing involved in B-thalassemia?

Answer 19

B-thalassemia can be caused by

• Point mutations in UAS –> decreased transcription
• Mutations in splice donor or splice acceptor sites –> aberrant splicing

Question 20

Systemic lupus Erythamatosis

Answer 20

Autoimmune disease where the body produces antibodies against its own components, including snRNPs, and triggers an inflammatory cascade –> multiorgan dysfunction

Question 21

Spliceosome is made up of

Answer 21

snRNPs: complexes of snRNAs (U1,U2,U4,U5,U6) + proteins

Question 22

What cuases myotonic dystrophy?

Answer 22

Expansion of CTG triplet repeats in the 3’UTR of the MDPK gene, which are transcribed into CUG repeats in mRNA, prevents nuclear export of the DMPK mRNA

The more repeats, the more severe

Question 23

How are improper nuclear export of mRNA and splicing involved in myotonic dystrophy?

Answer 23

1. As CUG repeats in the 3’UTR of the DMPK gene increase and can’t be exported, they build up in the nucleus and attract muscleblind.
2. Since muscleblind is now bound to DMPK mRNA, it isn’t binding and acting as an inhibitor to the CUG-BP mRNA

• –> Increased expression of CUG-BP protein , which is involved in embryonic splicing
• –> increased aberrant splice forms of cardiac torponin T, insulin receptor, and Cl- channel –> abnormal thenar reflex, insulin resistance, myotonia

Question 24

Where is the stop codon?

Answer 24

in the 3’-UTR. UAA

Question 25

When does hnRNA get processed (5’cap, 3’polyA tail, splicing) into mRNA?

Answer 25

Simultaneous with transcription