Exam 2- Transcription

Question 1

What are the different types of rRNA present in prokaryotes and eukaryotes?

Answer 1

Prokaryotes: 28S, 16S, 5S rRNA’s
Eukaryotes: 28S, 18S, 5.8S, 5S

Question 2

Describe the RNA Polymerase subunits involved in prokaryotic mRNA synthesis

Answer 2

Prokaryotic RNA Polymerase has a core enzyme that is made up of 2alpha, 1beta, and 1beta’ subunits that posses 5’ —> 3’ polymerase activity and 1alpha subunit that functions as an initiator that recognizes the promoter sequence

Question 3

What are the different RNA polymerase of eukaryotes?

Answer 3

RNA polymerase I- rRNA
RNA Polymerase II- hnRNA (precursor for mRNA)
RNA Polymerase III- tRNA

Question 4

Promoter elements in eukaryotes

Answer 4

TATA Box
GC Box
CAAT Box

RNA polymerase II starts polymerization once the transcription is initiated by transcription factors that bind to promoter regions of the gene that consists of promoter elements

Question 5

Describe the transcription factors involved in eukaryotic TATA binding

Answer 5

TFIID: TATA box always recognizes TFIID

• TBP: TATA binding protein
• TAF’s: TATA Box activating factors
• TFIIA’s & TFIIB’s: bind RNA Polymerase II

RNA polymerase II binds to TFIIF which interacts with TFIIE and TFIIH

• TFIIH (helicase) unwinds DNA
• RNA Polymerase II starts transcription

Question 6

What is the protein factors that binds to GC box?

Answer 6

SP1

Question 7

What are enhancers?

Answer 7

Enhancers bind to specific transcription factors far (>1000 bp’s) away from the general transcription initiation. During transcription, they bend from a far away distance to interact with the transcriptional machinery

• can be upstream (5’) or downstream (3’)
• can be sense or antisense orientation

Question 8

Give 2 examples of enhancers

Answer 8

• NFKB1 (Nuclear Factor k B1): binds to enhancer region of immune response genes and regulates the inflammatory response
• PEP Carboxy Kinase (PEPCK): PEPCK contains an enhancer called cAMP response element (CRE) that binds to cAMP response element binding protein (CREBP). It also contains another enhancer called glucocorticoid response element (GRE) that binds to cortisol banding protein

Question 9

Describe 2 enhancer elements of the PEPCK gene and their respective binding proteins

Answer 9

CRE (cAMP response element) that binds to CREBP (cAMP response element binding protein)

GRE (glucocorticoid response element) that binds to cortisol binding protein

Question 10

What are euchromatins and heterochromatic should?

Answer 10

Euchromatins- heavily acetylated making it transcriptionally activated part of the chromosome

Heterochromatins- hypermethylated making it transcriptionally inactive part of the chromosome

Question 11

How does methylation and acetylation affect gene transcription?

Answer 11

Acetylation: transcriptionally active
Methylation: transcriptionally inactive

Question 12

What is primary (pri-RNA/hnRNA)?

Answer 12

mRNA precursor synthesized by RNA Polymerase II

Question 13

Describe the 5’ and 3’ modifications that happens to primary RNA/hnRNA?

Answer 13

7meG-cap: 7-methyl guanine cap containing nucleotide is added to hnRNA at the 5’ end

• the cap itself would contain a 5’-5’ linkage (Me-G-P-P-P-5’-N) that protects the newly synthesized RNA from degradation
• In addition, the 7-meG-cap helps recognize the 5’ end of the processed mRNA by the ribosome complex during protein synthesis (ie translation initiation)

Poly-A tail: 3’ modification immediately after Tx of the complete hnRNA in the nucleus, a protein complex along with polyA polymerase (PAP) recognize the motif AAUAAA present upstream to the 3’ end of the hnRNA. PAP complex removes the hnRNA from RNA Polymerase II, before adding around 200 adenylyl residues at the 3’ end. The 3’ end with a bunch of A residues is called the polyA tail

Question 14

What is the recognition motif needed in the hnRNA for it to be poly adenylated.

Answer 14

AAUAAA

Question 15

What are some purported functions for the poly A tail

Answer 15

Facilitation of hnRNA transport from nucleus to cytosol

Stability of mRNA

Question 16

Describe the process of splicing.

Answer 16

Exon1-Intron1 junction has the motif GpGU
In the middle of the intron, the prominent catalytic A called branch site A
At the intron1-exon2 junction there is a consensus motif AGp

First 2’-OH of the branch site A attacks the phosphodiester of G on the GpGU site
Now the cleaver exon 1 attaches the 5’ end of the exon 2 and form spliced Exon1-Exon2

Splicing requires small nuclear ribonucleoprotein particles (SnRNP’s)

Question 17

What is mRNA copy number?

Answer 17

Transcripts per cell

Question 18

Describe the processing of tRNA.

Answer 18

For tRNA, 5’ leader sequences removed first
Introns are removed and some bases are modified
At the 3’ end, UU are removed and the 3 bases CCA are added
The CCA at the 3’ end of the tRNA is crucial for amino acid addition (ie charging) during translation

Question 19

Describe the processing of hnRNA.

Answer 19

Splicing - snRNA in the splicing of exons and introns on hnRNA to obtain mature mRNA

5’cap
3’PolyA tail

Question 20

Describe the mode of actions of rifampicin, actinomycin D,k and alpha-amanitin

Answer 20

Rifampicin: specific to Beta-subunit of prokaryotic RNA polymerase
- Abx

Actinomycin D: binds to DNA template and interferes with the movement of RNA polymerase along the DNA
- Chemotheurapeutic

Alpha-amanitin: it is a peptide produced by the mushroom Amanita phalloides. It inhibits eukaryotic RNA polymerase II. Liver toxicity is the most immediate effect
- toxin

Question 21

Describe the transcriptional regulation of p21.

Answer 21

p21: cyclin dependent kinase inhibitor
P53 (guardian of the genome) is an anti-oncogene protein product that binds to the promoter of the p21 gene and causes upregulation of p21 gene product which in turn stops cell cycle progression by inhibiting cell cycle kinases

Question 22

Describe the transcriptional regulation of gamma-globulin

Answer 22

GATA-1 is a suppressor of gamma globulin gene. GATA-1 keeps the mRNA expression at an appropriate amount so that the proper stoichiometry of alpha and beta Hb chains can be maintained.

Upregulation of gamma-globulin leads to down-regulation of beta-globulin

Question 23

Thalassemia

Answer 23

Autosomal disorder
G to A mutation of upstream element of gamma Hb gene results in loss of binding by transcription factor, GATA-1
Upregulation of gamma-globin, down regulation of beta-globin