Transcription

Question 1

Transcription is ___-directional and synthesizes nucleotides in a __—->__ fashion.

Answer 1

uni-directional; 5’ –> 3’

Question 2

What are the five basic sterps of transcription?

Answer 2

1. RNA Pol binds the promoter on closed DNA
2. RNA Pol melts DNA forming transcription bubble, aka open complex
3. Pol catalyzes linkage of first two rNTPs
4. Pol advances down DNA 3’->5’ linking and melting
5. Pol releases RNA and falls off DNA at transcription stop site

Question 3

Describe the 4 cellular RNA polymerases

Answer 3

Bacterial RNA Pol: does everything
Human Pol I: rRNA
Pol II: mRNA, snRNA, miRNA. It has CTD!!!
Pol III: tRNA

Question 4

Define a promoter and give an example

Answer 4

Sequence upstream of a gene that positively impacts transcription. TATA box 30bp upstream

Question 5

How does alpha-amanitin work?

Answer 5

It inhibits the movement of RNA Pol II, binding its bridge substructure so that translocation of the polymerase down the DNA chain can’t happen.

Question 6

How does rifampicin work?

Answer 6

Binds the beta subunit of bacterial RNA Pol; plugs up the exit chamber where assembled RNA exits the transcriptional complex.

Question 7

Describe structures of the Pre-Initiation Complex.

Answer 7

TFII-A,B,D,E,F,H, Pol II, and Med
TFIID = TBP (TATA Binding Protein)
TFIIH = Helicase and phosphorylates CTD

Question 8

Describe the clinical syndromes caused by mutations in TFIIH subunits.

Answer 8

Problems with NER: Cockayne’s syndrome, Trichothiodystrophy, Xeroderma pigmentosum - light sensitivity, abnormal pigmentation, cancer susceptibility, neuorological abnormalities, unscheduled DNA synthesis

Question 9

Describe the three major ways in which most pre-mRNA’s are processed.

Answer 9

1. The first 5’ triphosphate is replaced with a backwards, 7-methylguanosine
2. Splicing: excision of introns and desegmentation of exons
3. Cleavage/Polyadenylation: cleavage of RNA at 3’ end past the consensus sequence and polyadenylation (> 200 A’s) of cleaved site.

Question 10

List the 3 functions of the 5’ cap of the mRNA.

Answer 10

1. Prevents degradation by exonucleases (removal signals degradation)
2. Facilitates splicing & processing via CBC
3. Recognized by eIF4E for transport to ribosomes

Question 11

List the three reactions required to add a 5’ cap to pre-mRNA

Answer 11

1. Cut off PO4 at 5’ end
2. Add GTP backwards via Guanylyl Transferase (It becomes GMP). Keep 2 PO4 from mRNA
3. Methylate the 7-position of guanosine cap via SAM. Thus, 7-methyl-guanosine cap

Question 12

What conserved sequences are at the 5’ and 3’ ends of most introns?

Answer 12

Splice site at 5’ end of intron: GU (beginning)

Splice site at 3’ end of intron: AG. (last 2 bases)

Question 13

What is the consensus sequence at poly-A site?

Answer 13

AAUAAA

Question 14

Describe how alternative splicing permits multiple proteins to be produced by splicing defects.

Answer 14

Remove/retain certain exons
mutually exclusive exons
exon truncation or extension at 5' end
exon truncation or extension at 3' end
intron retained

Question 15

How can alternative splicing go awry?

Answer 15

The 5’ splice site at any intron is still part of the DNA, and is therefore vulnerable to mutation

Question 16

Provide two examples of genetic disorders caused by splicing defects.

Answer 16

Marfan’s syndrome: disruption of splicing the fibrilin gene transcript (fibriling = conn. tissue in wall of heart and blood vessels). Tall people prone to aneurysms.
CD44: abnormal splicing is a predictor of tumor metastasis (diag. & prog. marker)

Question 17

Describe the function of U1 and U2 snRNA’s in splicing

Answer 17

U1: binds GU
U2: binds branch point (A)
U2AF: binds AG
Lariat mechanism where U1 brings 5’ into proximity with branch point. U2 activates the 2’ OH at branch point, which attacks the phosphodiester bond just past GU. Now, the free 3’ OH on the end of the 5’ exon attacks the phosphodiester bond at the AG 3’ splice site, linking the two exons and excising the snRNP/intron complex to be degraded

Question 18

Where are the 5’ and 3’ UTR sequences

Answer 18

5’: between +1 and first Start Codon (XUG)

3’: after stop codon (UAA, UAG, UGA) through poly-A tail

Question 19

Describe the two reactions that make the mature 3’ end of mRNA’s.

Answer 19

1. Recognition and cleavage of pre-mRNA after AAUAAA

2. Polyadenylation of 3’ end

Question 20

The poly(A) tail is important for what 3 things?

Answer 20

nuclear export, translation, and stability of mRNA.

Question 21

What event terminates transcript?

Answer 21

3’ end processing (cleavage/polyadenylation)

Question 22

Provide an example of how alternative poly A sites can be used to make more than one protein from a single gene.

Answer 22

Just like alt. splicing, it depends on where the poly A is added. Example: two forms of IgM (memb-bound and secreted) are formed by alt poly-A sites in their common gene.