Transcription

Question 1

How are phosphodiester bonds formed by RNA polymerase?

Answer 1

3’OH attacks alpha phosphate attached to 5’ carbon (releases 2 P groups)

Question 2

5 steps in transcription cycle

Initiation, Elongation, Termination

Answer 2

1) RNA pol binds promoter sequence on helical DNA in a closed complex
2) Pol melts DNA strands apart near transcription start site forming an open complex
3) Polymerase catalyzes phosphodiester linkage of two initial rNTPs
4) Polymerase advances 3’–> 5’ down template strand, linking rNTPs 5’–>3’
5) At transcription STOP site, polymerase releases completed RNA and dissociates from DNA

Question 3

RNA pol I

Answer 3

makes rRNA

Question 4

RNA pol II

Answer 4

• makes mRNA, snRNA, microRNA
• CTD
• Needs general transcription factors (TFIIH, TFIID)
• Uses sliding clamp

Question 5

C-terminal Domain (CTD)

Answer 5

• on RNA pol II

- acts as a landing pad for proteins that effect elongation/processing

Question 6

RNA pol III

Answer 6

makes tRNA

Question 7

Promoter

Answer 7

• sequence of DNA upstream of the transcription start site that direct RNA polymerase to the start of genes
• controls FREQUENCY of transcription
• site of pre-initiation complex assembly

Question 8

TATA Box

Answer 8

• promoter sequence (TATAAA)
• 30 bases upstream from start site
• conserved
• site where GTFs bind

Question 9

TATA box binding protein

Answer 9

• binds TATA box

- directs assembly of pre-initiation complex of general transcription factors at the promoter

Question 10

+1 base

-) and (+

Answer 10

-first base pair where transcription initiates

(-) = upstream
(+) = downstream

Question 11

Proximal Promoter Elements

Answer 11

• gene specific sequence that specific TFs bind

- Influence activity of the promoter

Question 12

Enhancer

Answer 12

• either up or down stream
• can be very far away
• bind transcriptional activator proteins and effect transcription
• cell type specific

Question 13

a-amanitin (and transcription)

Answer 13

• non-competitive inhibitor of RNA polymerase

- Binds bridge helix of RNA pol II blocking translocation

Question 14

Rifampicin (and transcription)

Answer 14

• Inserts itself into RNA exit channel of bacterial RNA polymerase
• Block growth of the RNA chain

Question 15

TFIID

Answer 15

• part of RNA polymerase II pre-initiation complex
• aka TATA binding protein
• promoter recognition GTF
• binds TATA box on DNA sequence

Question 16

TFIIH

Answer 16

• part of RNA polymerase II pre-initiation complex
• promoter recognition GTF
• Functions in transcription and DNA repair (nucleotide excision)
• has multiple subunits (CDK7 protein kinase and XPB helicase)

Question 17

CDK7 Protein Kinase

Answer 17

• part of TFIIH
• phosphorylates RNA pol II CTD during promoter clearance
• Allows pol II to elongate

Question 18

XPB helicase

Answer 18

• part of TFIIH

- acts as helicase to open up DNA strands

Question 19

Syndromes associated with mutations in TFIIH (3)

Answer 19

• Xeroderma Pigmentosum (XP)
• Cockayne’s Syndrome (CS)
• Trichothiodystrophy (TTD)

Question 20

Xeroderma Pigmentosum (XP)

Answer 20

mutation in TFIIH so that is is unable to carry out nucleotide excision repair for damage caused by UV light

Question 21

3 ways pre-mRNAs are processed

Answer 21

1. Capping
2. Splicing
3. Cleavage/Polyadenylation

Question 22

Capping

Answer 22

replacement of 5’triphosphate from the first rNTP with a backwards 7-methylguanosine (no phosphate group)

Question 23

Splicing

Answer 23

excision of introns, desegmentation of exons

Question 24

Cleavage/Polyadenylation

Answer 24

cleavage of RNA at 3’end past consensus sequence, and polyadenylation (addition of > 200A’s) of cleaved site

Question 25

Pre-mRNA

Answer 25

- initial product of transcription that must be extensively processed into a mature form (mRNA) - has introns AND exons - no cap, no tail

Question 26

Processing into mRNA enhances _________, _________, and ___________

Answer 26

nuclear export, translation, preventing degredation

Question 27

Function of 5'cap of mRNA (4)

Answer 27

1. Protects the 5'end from exonucleases 2. Recognized by cap-binding proteins - crucial for splicing/processing 3. Recognized by TF eIF4E for nuclear export into cytoplasm 4. Removal of cap signals mRNA degredation

Question 28

3 reactions required to add a 5' cap to pre-mRNA

Answer 28

1) Triphosphatase - removes gamma phosphate at end of primary transcript 2) Guanylytransferase - synthesizes 5'-->5' phosphodiester linkage 3) Guanine 7-methyltransferase - addition of methyl group at the 7 position

Question 29

Splice site at 5' end of intron

Answer 29

GU

Question 30

Splice site at 3' end of intron

Answer 30

AG

Question 31

Consensus sequence at poly-A site

Answer 31

AAUAAA

Question 32

Alternative splicing

Answer 32

allows many different proteins to be encoded by a single gene

Question 33

Genetic disorders caused by splicing defects (2)

Answer 33

1) CD44 - tumor metastasis | 2) Spinal muscular atrophy

Question 34

CD44 and tumor metastasis

Answer 34

abnormal splicing of CD44 contributes to tumor metastasis

Question 35

Spinal muscular atrophy treatment

Answer 35

can activate alternate SMN2 gene to take over for defective SMN1 gene by using alternative splicing

Question 36

HIV and alternative splicing

Answer 36

HIV uses alternative splicing to generate many more mRNAs from a single transcript

Question 37

Steps of splicing (4) (trans-esterification reactions)

Answer 37

1) U2snRNA base pairs with branch point A, and activates it 2) U1 snRNA basepairs with 5' splice site 3) 2'OH of branch point A then attacks phosphodiester bond IN FRONT of GU marker ath beginning of intron 4) 3'OH of exon 1 attacks phosphodiester bond at AG splice site, linking exon 1 and exon 2

Question 38

U2 snRNA

Answer 38

- key role in splicing reactions | - base pairs with branch point A and activates it

Question 39

U1 snRNA

Answer 39

- key role in splicing reactions | - base pairs with 5' splice site

Question 40

Transcription start site

Answer 40

+1 position

Question 41

Introns

Answer 41

non-coding sequences between GU (5') and AG (3')

Question 42

branch point

Answer 42

- single A residue - usually in intron between GU (5') and AG (3') - base pairs with U2 snRNA (**VITAL part in first step of splicing**)

Question 43

Exons

Answer 43

coding sequences

Question 44

5'UTR

Answer 44

- untranslated region - between +1 and start codon on the processed mRNA strand - located at beginning of first exon

Question 45

3'UTR

Answer 45

- untranslated region - located after stop codon, at the end of the last exon - contains highly conserved polyA site

Question 46

initiation/start codon

Answer 46

- 5'AUG | - encodes methionine

Question 47

termination/stop codon

Answer 47

3' UAG, UAA, UGA

Question 48

polyA site

Answer 48

consensus sequence AAUAAA

Question 49

Two reactions that make mature 3' end of mRNAs

Answer 49

1) Cleavage: recognize consensus sequence (AAUAAA) at 3' end of pre-mRNA and cleave 2) Polyadenylation: polyadenylation of free 3' hydroxyl group

Question 50

3' end processing of pre-mRNA is coupled with ________________ because ____________

Answer 50

- termination of transcription by RNA pol II | - because 3' polyA tail necessary for RNA polymerase complex to detach from the DNA

Question 51

Function of mRNA polyA tail (2)

Answer 51

1) protection from degradation | 2) Export of mRNA from the nucleus

Question 52

Mutations in polyA site can result in _________

Answer 52

Thallasemia

Question 53

Thallasemia

Answer 53

results from mutations of the AAUAAA poly A site consensus sequence in globin genes

Question 54

IgM (immunoglobulin M) and alternative poly A sites

Answer 54

Membrane bound and secreted forms are formed based on alternative polyA sites in their common genome

Question 55

Cancer cells and alternative poly A sites

Answer 55

use altered polyA site choice to shorten the 3'UTR region, allowing them to escape regulation by microRNAs