Molecular Biology 7 : Transcription in eukaryotes

Question 1

What is the role of the general transcription factors ?

Answer 1

To position RNA polymerase at the start site.

Question 2

What happens during the formation of the preinitiation complex (PIC) ?

Answer 2

TFIIH phosphorylated the polymerase C- terminal domain (CTD), leading to melting of the DNA to form a transcription bubble.

Question 3

OF all TFs at the PIC, which one stays attached to RNA Pol ?

Answer 3

TFIIF (stabilizes the polymerase)

Question 4

In which order do the different factor bind the RNA Pol II in eukaryotic initiation of transcription ?

Answer 4

• TBP and TFIID bind the TATA box
• TFIIA and TFIIB are recruited
• RNA Pol II, already bound to TFIIF, binds the promoter region
• TFIIE and TFIIH are finally recruited
• TFIIH phosphorylates the CTD of RNA Pol II, which triggers initiation of elongation

Question 5

What kind of 5’ end does the newly transcribed RNA have ?

Answer 5

pppA

Question 6

What is the structure of the 5’ methylated cap (7-methyl guanylate) of eukaryotic mRNA ?

Answer 6

• 5’-5’ linkage
• 7-methyl-guanylate
• Methyl group on the 2’ hydroxyl of the ribose of the first mRNA nucleotide (sometimes)

Question 7

What are the 4 steps in the addition of the 5’-methyl cap to nascent mRNA ?

Answer 7

1. Phosphatase: removal of the gamma-phosphate group at the 5’ end of the precursor.
2. The 5’ end receives a GMP group from GTP in a reaction catalyzed by guanyltransferase.
3. The base of the guanylate group is methylated at N-7.
4. The 2’-hydroxyl groups of the terminal and the penultimate ribose groups of the precursor may also be methylated.

Question 8

What is the consequence of the phosphorylation of Pol II CTD ?

Answer 8

Phosphorylation of PolII CTD leads to an exchange of initiation factors for factors involved in elongation and RNA processing.

Question 9

What are the general characteristics of transcription in eukaryotes ?

Answer 9

• Closely linked to processing of mRNA
• Termination signal sequence at the end of the gene triggers transfer of polyadenylation enzymes to the RNA.
• Mature RNA released, remaining RNA associated with Polymerase degraded.
• Polymerase doesn’t terminate immediately-second uncapped transcript.

Question 10

How is does polyadenylation occur in termination of transcription ?

Answer 10

• RNA Pol II reaches and trancribes the poly-A signal sequence in DNA
• the ply-A signal sequence in mRNA is bound by CstF (Cleavage stimulating Factor) and CPSF (Cleavage and Polyadenylation Specificity Factor)
• the RNA is cleave at the poly-A site and CstF dissociates
• PAP (Poly-A-Polymerase) binds to the 3’ end of RNA and extends the 3’ ends w/ a poly-A tail
• poly-A-binding proteins bind the poly-A tail as it is formed
• when poly-A tail is complete (~200 As), CPSF and PAP dissociate

Question 11

What are the 4 steps of mRNA processing in eukaryotes ?

Answer 11

1. Transcription, 5’capping
2. Cleavage at Poly(A) site (endonuclease)
3. Polyadenylation (PAP + ATP)
4. RNA splicing

Question 12

How many exons does human DNA ligase contain ? Spanning what length ?
What about human BRCA1 ?

Answer 12

Human DNA ligase : 28 exons, spanning 53 kb

Human BRCA1 : 24 exons of BRCA1 spanning 81 kb

Question 13

Why did Richard J. Roberts and Phillip A. Sharp win the the Nobel Prize in Physiology or Medicine in 1993 ?

Answer 13

For their work on “Split genes and RNA Splicing”.

Question 14

What are the RNA splicing signals ?

Answer 14

5’ splice site : AG/GU(AorG)AGU
/ = exon/intron boundary
Branch site = A (in the very middle of the intron)
3’ splice site: [Py tract]AG/G

Question 15

What is the splicing reaction ?

Answer 15

2 transesterification reactions :

• the 2’ OH of the A branch site attacks the 5’ phosphate and binds it
• the free 3’ OH now attacks phosphate on the 3’ splice site, detached the spliced exons from the intron lariat

Question 16

What is the spliceosome ?

Answer 16

• Molecular “machine” similar in size to a ribosome
• Comprises about 150 proteins and 5RNAs called small nuclear RNAs (U1, U2, U4, U5 and U6)
• Each RNA is complexed with several proteins (snRNPs).
• Hydrolyses ATP

Question 17

Why are RNA-RNA hybrids formed during splicing ?

Answer 17

The snRNAs form RNA-RNA hybrids w/ the spliced RNA.:

• U1 : CAUUCA
• U2 : AUGAUG
• U6 : UGAUCAGC

Question 18

Why does alternative splicing expands the informational content of the genome ?
How can this be implicated in disease ?

Answer 18

• At least 45-60 % of human genes produce primary transcripts that can be alternatively spliced to generate a great number of potential mRNAs and protein isoforms.
• Up to 40% of genetic defects can be associated with incorrect RNA processing.

Question 19

What are calcitonin (CT) and calcitonin gene-related peptide (CGRP) ?

Answer 19

• Two peptide hormones from same gene :
- CT expressed in the thyroid
- CGRP expressed in brain.
• CT (a 32-amino-acid peptide) is a hormone primarily involved in protecting the skeleton during periods of “calcium stress”.
• CGRP (a 37-amino-acid neuropeptide) is the most potent endogenous vasodilatory peptide.

Question 20

Why are calcitonin (CT) and calcitonin gene-related peptide (CGRP) a good example of alternative splicing (tissue specific RNA processing) ?

Answer 20

Alternative splicing involves use of alternate polyA sites –> alternative use of 3’ end exons.
Exons 1-3 = constitutive.
Exon 4 = used in thyroid cells.
Exons 5 & 6 = used in brain tissue.
Post-transcriptional processing of protein also occurs to generate mature peptides.