Transcription

Question 1

Who mRNA is modified and how?

Answer 1

Eukaryotes

1) 5’ cap
2) Polyadenylation of 3’ end
3) Splicing of exons and removing exons

-RNA polymerase II does the job

(Prokaryotes are unmodified)

Question 2

Capping 5’ enzymes

Answer 2

1) Phosphatase
2) Guanylyl transferase
3) Guanine-7-methyl transferase
4) 2’-O-methyl transferase

Function of cap:

1) Processing
2) Transport
3) Translation
- Increases half-life, prevent degradation, helps export

Question 3

Modifications of the 3’ tail

Answer 3

Cleavage:
1) CPSF binds to hexamer AAUAAA
2) CstF binds to GU-rich element near by
3) Cleavage factors bind to CA sequence at the cleavage site = producing nucleotides
(eventually cleaved fragments of RNA degraded in nucleus)
4) Poly-A-Polymerase adds nucleotides to 3’
5) Poly-A-Bindnig proteins (PABP) binds to poly-A tail = Assists in directing translation by the ribosome

Functions of tail:

1) increases half-life
2) Protects from degradation in cytoplasm
3) Protects mRNA from ribonuclease attack (PABP binds to poly(A)
4) Aids in transcription termination
5) Export of the mRNA from nucleusbj

Question 4

Splicing DNA and RNA

-Donor sites/acceptors

Answer 4

DNA: (Intron splicing site)
Donor Site DNA: GT
Acceptor Site DNA: AG

RNA: (Intron splicing site)
Donor Site RNA: GU
Acceptor Site: AG

-Each splicing event removes 1 intron

Question 5

Intron removal from pre-mRNA

Answer 5

5 snRNP’s that mediate splicing

1) U1
2) U2
3) U4
4) U5
5) U6

Process:

• When U1 and U4 leaves = splicesome formed
• When U2, U5, and U6 = alignment, allowing two transesterification rxns to occur
• 2-OH of Site A attacks the 5’ phosphate =lariat structure, leaves free 3’-OH
• 3’-OH attacks 5’-phosphate at splice-acceptor to join exon
• Intron released as lariat=degrades
• Mature messenger RNA molecules pass into the cytosol through pores in nuclear membrane

Question 6

Lupus

Answer 6

• Spliceosome disease
• Autoimmune

Symptoms:

• Fatigue
• Arthritis
• Fever
• Skin rashes
• Kidney problems

Question 7

Alternative splicing

Answer 7

RNA can be spliced in different ways to produced different species of mRNA

-Can repress or activate splicing at site

(Increases diversity of of mRNA and expressed proteins)

Question 8

B-thalassemia

Answer 8

• Spiceosome disease

- Mutation for B-Globin gene, it generates additional splice sites within mRNA

Question 9

Limb Gridle Muscular Dystrophy

Answer 9

• Mutation in the calpain-3-gene that generates a new splice site within EXON 16 (makes shorter)
• makes defective protein

Question 10

RNA editing

Answer 10

Two types:
1) Adenosine to Inosine (A–>I): Alters Ca+ permeability, required for brain development

2) Cytidine to Uridine (C–>U): Forms premature stop codon
- (Increases diversity of of mRNA and expressed proteins)

Question 11

Post transcriptional modifications

1) Zymogens
2) Protein Phosphorylation by kinase
3) Glycosylation
4) Lipid Anchoring
* Proteolytic processing of Insulin

Answer 11

1) Zymogens:
- Activates inactive enzymes

2) Protein Phosphorylation by kinase:
- Phosphate transferred from ATP to Amino side chain

3) Glycosylation:
- Alters the properties of proteins, changing their stability, and physical bulk
- O-Linked glycosylation: Carbohydrate chain attached to OH group of Ser/Thy
- N-Linked glycosylation: Carbohydrate chains attacked to the amide of nitrogen Asn residue

4) Lipid Anchoring:
- Targets RA proteins to the cystolic (inner leaflet) by lipid anchoring method

Proteolytic processing of Insulin
-Insulin and C-peptide packed together into secretory vesicles for secretion

Question 12

Types of RNA

Answer 12

1) mRNA-(messenger RNA), transcripts of protein coding genes
2) tRNA- (transfer RNA), brings amino acids to the ribosome
3) rRNA- (ribosomal RNA)- combines ribosomal proteins to form ribosome
4) snRNA-(Small nuclear RNA) -combines certain proteins and gene regulation

Question 13

What are the different RNA polymerase’s

• Prokaryotes
• Eukaryote

Answer 13

Prokaryotes:
-Sigma factor

Eukaryotes:

1) RNA polymerase I: rRNA genes
2) SNA polymerase II: all coding genes (mRNA/snRNA)
3) RNA polymerase III: tRNA genes/some snRNA

Question 14

What are the different promoters?

• Prokaryotes
• Eukaryote

Answer 14

Prokaryotes:

• Pribnow Box
• 35–>-10 Bp upstream from start site

Eukaryote:
-TATA box, GC box, CAAT box

Question 15

Transcription of Prokaryotes

Answer 15

1) Binding of RNA Polymerase to promoter (Pribnow box)
2) DNA unwinds
3) RNA transcribes using antisense template
4) Separation of sigma factor and RNA polymerase
5) RNA polymerase continues to unwind DNA and code
6) Transcription is terminated w/ terminated sequence

-GYRASE relieves positive supercoiling strain by placing negative supercoiling behind DNA polymerase

Question 16

Transcription of eukaryotes

Answer 16

1) TATA binding protein (TBP), and TFIID bind to promoter at TATA box
2) TFIIB binds to promoter
3) RNA polymerase, TFIIE, and TFIIH recruited to promoter
4) TFIIH unwinds DNA w helicase
5) TFIIH activates RNA polymerase, then transcription begins
6) Transcriptions factors released
- Topoisomerase I relieves positive supercoiling strain by placing negative supercoiling behind DNA polymerase

Question 17

Transcription

Intrinsic Termination

Extrinsic Termination

Answer 17

Intrinsic Termination:

• Palindromic sequences
• Includes stem loop (G-C rich regions and uracil) = Termination structure
• Termination structure recognized by RNA polymerase = RNA dissociates from DNA/RNA

Extrinsic Termination:

• Rho protein assists in release of RNA polymerase
• Termination sequence creates loop structure (termination structure), once cut it unwinds