Lecture 8 - Transcription 2

Question 1

When does transcription and translation happen? (Pro vs. Eu)

Answer 1

Prokaryotes: At the same time in the cytoplasm
Eukaryotes: Transcription in the nucleus then translation in the cytoplasm

Question 2

Gene structure (Pro vs. Eu)

Answer 2

Prokaryotes: DNA sequence is read in the same order as the amino acid sequence
Eukaryotes: Noncoding introns within coding sequence

Question 3

Modification of mRNA after initial transcription but before translation (Pro vs. Eu)

Answer 3

Prokaryotes: None
Eukaryotes: Introns spliced out; 5’ cap and 3’ poly A tail added

Question 4

Locations of functionally related genes (Pro vs. Eu)

Answer 4

Prokaryotes: Often clustered in operons
Eukaryotes: Often distant from one another with separate promoters

Question 5

RNA polymerases (Pro vs. Eu)

Answer 5

Prokaryotes: One
Eukaryotes: Three: I transcribes rRNA, II transcribes mRNA, III transcribes tRNA and small RNAs

Question 6

Promoters and other regulatory sequences (Pro vs. Eu)

Answer 6

Prokaryotes: Few
Eukaryotes: Many

Question 7

Initiation of transcription (Pro vs. Eu)

Answer 7

Prokaryotes: Binding of RNA polymerase to promoter (Holo RNA polymerase)
Eukaryotes: Binding of many proteins, including RNA polymerase [General (or basal) transcription factors]

Question 8

Core promoter

Answer 8

Recognized by general (basal) transcription factor that recruit RNA polymerase

Question 9

Promoter proximal and distal elements

Answer 9

Regulatory sequences and binding sites for transcriptional activators and repressors

Question 10

TATA box

Answer 10

(So called because it is rich in AT base pairs), where DNA begins to denature so that the template strand can be exposed. Normally located 25 nucleotides upstream from the transcription start site

Question 11

Transcription factors

Answer 11

Proteins that assemble on a eukaryotic chromosome, allowing RNA polymerase II to perform transcription

Question 12

Initiation

Answer 12

1. The first transcription factor, TFIID, binds to the promoter at the TATA box. (subunit of TFIID that recognises it is TBP = TATA Binding Protein)
2. This attracts the binding of other transcription factors to form a transcription initiation complex (like TFIIB which is critical for the recruitment of RNA polymerase II)
3. RNA polymerase II binds
4. More transcription factors are added (TFIIE and TFIIH)
5. RNA polymerase is ready to transcribe RNA

Question 13

TFIIH

Answer 13

• Has 9 subunits one being DNA helicase, which helps RNA polymerase gain access to the template strands at the transcription start
• Adds phosphate groups to the ‘tail’ of RNA polymerase (known as the CTD or C-terminal domain) causing conformational changes and allowing it to move away from the promoter and enter elongation phase

Question 14

Which factors carry out functions similar to that of sigma factors in bacteria?

Answer 14

General transcription factors

Question 15

General Transcription Factors

Answer 15

Called ‘general’ because they are added to nearly all promoters used by RNA polymerase II

Question 16

Explain 5’ capping in mRNA

Answer 16

1. A phosphatase removes a phosphate from the 5’ end of the nascent RNA
2. A guanyl transferase adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’) [G cap]
3. A methyl transferase adds a methyl group to the guanosine
   These three enzymes modify the 5’ end of the nascent transcript as soon as it emerges from the polymerase

Question 17

How does the 5’ cap help?

Answer 17

1. Facilitates mRNA binding to the ribosome

2. Protects RNA from ribonucleases

Question 18

Explain cleavage and polyadenylation of the 3’ end of mRNA

Answer 18

1. AAUAAA sequence (polyadenylation) signal in the pre-mRNA (after last codon) signals an endonuclease to cut
2. Poly(A) polymerase adds 100 to 300 adenines

Question 19

How does the poly A tail help?

Answer 19

1. May assist in export from nucleus

2. Important for stability of mRNA an the efficient translation

Question 20

pre-mRNA splicing

Answer 20

1. Small nuclear ribonucleoprotein (snRNP) bind to consensus sequences near the 5’ and 3’ splice sites (these normally begin with GU at the 5’ and ends with AG at its 3’ end)
2. Interactions between the two snRNPs and other proteins form a spliceosome
3. Cut is made between the 5’ exon and the intron
4. Intron forms a closed loop, like a lariat
5. Free 3’ OH group at the end of the cut exon reacts with the 5’ phosphate of the other exon
6. 3’ exon is claved and spliced to the 5’ exon
7. mature mRNA is exported for translation
8. excised intron is degraded in nucleus

Question 21

Alternative splicing

Answer 21

• Can give more than one product from a single gene

- Can be regulated to give differential gene expression

Question 22

How is mRNA exported from the nucleus to cytoplasm?

Answer 22

1. Mature mRNA leaves the nucleus through nuclear pores
2. Proteins bind tot he 5’ end that are recognized by receptors at the nuclear pore
3. These proteins lead the mRNA through the pore