Lecture 36: RNA Structure, Synthesis, Processing

Question 1

Transcription features

Answer 1

• DNA read 3’ → 5’
• RNA synth. 5’ → 3’
• No primer req.
• Coding (sense) strand = 5’ → 3’; copy of RNA
• Template (non-coding) strand = 3’ → 5’; read to RNA

Question 2

Bacterial RNA polymerase structure

Answer 2

β subunit = catalytic site making 3’,5’ phosphodiester bonds
σ subunit = recognizes promoters to init. transcription
α subunit = structural
β’ subunit = binds DNA template
Core enzyme = α2ββ’
Holoenzyme = α2ββ’σ

Question 3

RNA vs DNA

Answer 3

• RNA has ribose (not deoxyribose)
• RNA Uracil not Thymine
• RNA shorter than DNA
• RNA always ss
• RNA forms secondary loop + stem structures

Question 4

3 steps of RNA polymerase transcription process

Answer 4

1. Initiation
2. Elongation
3. Termination

Question 5

Prokaryotic promoter features

Answer 5

• Upstream of transcription init. start site
• Written on sense strand
• Recognized by σ subunit

Question 6

Transcription initiation process

Answer 6

1. σ binds specific DNA promoters
2. Transcription bubble opens (always 12 bp)
3. σ dissociates as elongation starts w/ complementary bases

Question 7

Specificity of RNA polymerase σ binding to promoters

Answer 7

• Distance between -35 and -10 sites critical; must align w/ σ
• σ DNA binding domain unmasked when bound to Core enzyme
• Core binds randomly to DNA, σ binds core (if not at promoter, σ dissociates)

Question 8

RNA polymerase β DNA binding

Answer 8

2 nucleotide binding sites on β subunit
1. First 5’ end purine (1st P-diester bond)
2. All subsequent nucleotides

Question 9

Transcription elongation process

Answer 9

1. RNA-DNA hybrid helix ~8 bases long forms
2. σ released as 12 bp transcription bubble proceeds down DNA

Question 10

Transcription termination mechanisms

Answer 10

1. Rho independent
2. Rho dependent

Question 11

Rho independent transcription termination

Answer 11

Requires GC-rich palindrome followed by UUUs
1. Palindrome folds to hairpin followed by string of UUUs
2. Polymerase slows down
3. Weak string of A-U bonds cause transcript release

Question 12

Rho dependent transcription initiation process

Answer 12

ρ = helicase + ATPase
1. ρ binds RNA transcript
2. Polymerase slows down at GC-rich pre-termination seq.
3. ρ moves to 3’ end of transcript
4. ρ helicase unwinds RNA/DNA hybrid
5. ρ + RNA polym. dissociate from RNA

Question 13

How does rifampin affect transcription?

Answer 13

Inhibits formation of 1st phosphodiester bond during initiation (TB treatment), binds β subunit

Question 14

How does actinomycin D affect transcription?

Answer 14

Intercalates between DNA strands in major groove to inhibit initiation + elongation; prevents DNA strand unwinding.
Effective in prokaryotes and eukaryotes.
Would also inhib. DNA synthesis.

Question 15

Eukaryotic vs prokaryotic transcription (RNA synthesis)

Answer 15

Eukaryotes:
1. 3 polymerases, for rRNA, tRNA, sRNA
2. No σ subunit
3. Initiation by polym.-protein interaction, not polym.-DNA

Question 16

Eukaryote RNA polymerase I

Answer 16

Produces large rRNAs in nucleolus: 28S, 18S, 5.8S

Question 17

Eukaryote RNA polymerase III

Answer 17

Produces small rRNAs and tRNAs in nucleus: 5S, tRNA

Question 18

Eukaryote RNA polymerase II

Answer 18

Produces mRNA precursors: heterogenous nuclear (hnRNA) or primary transcripts, also miRNAs, snRNAs

Question 19

How is eukaryotic RNA polymerase II activity regulated?

Answer 19

RNA polym. II interacts w/ specific proteins which bind core promoter regulatory elements to control timing/location/context of expression

Question 20

RNA polymerase II Basal Transcription Complex assembly process

Answer 20

1. TBP (TATA binding protein)
2. Many TAFs (TATA assoc. factors)
3. Many other TFII transcription factors
4. Finally, RNA poly II

Question 21

Eukaryotic promoter/enhancer regions

Answer 21

+1/Inr (initiator)

Promoter located on sense strand
- Basal region
- Constitutive elements
- Inducible element

Enhancer anywhere, regulates transcription but not part of promoter

Question 22

RNA poly II core promoter basal element

Answer 22

Transcript. init. site (TATA box, -25)

Question 23

RNA poly II core promoter constitutive elements

Answer 23

Gene specific elements (can be many), control rate of transcript.

Question 24

RNA poly II core promoter inducible element

Answer 24

Specificity of transcript., i.e. environ. stimuli e.g. hormone response elements

Question 25

Phosphorylation control of RNA poly II initiation

Answer 25

One TFII transcription factor in the Basal Transcription Complex is a kinase; RNA poly II phosphorylation is req. for initiation.
De-Pi stops transcription.

Question 26

How do other proteins binding to promoter/enhancer regions affect transcription?

Answer 26

These other proteins change conformation of DNA, affecting efficiency of RNA poly II binding and transcription.

Question 27

Non-protein binding factors influencing RNA poly II transcription

Answer 27

Chromatin remodeling, histone acetylation

Question 28

How does α-amantin affect transcription?

Answer 28

Inhibits RNA polym. II (much more than poly III, not poly I) leading to liver failure and death

Question 29

How are RNA poly I and RNA poly III regulated?

Answer 29

Minimally

Question 30

RNA polymerase I transcription initiation process

Answer 30

1. Upstream + Core Binding Factors bind split promoter
2. RNA poly I binds

Question 31

RNA polymerase III transcription initiation process

Answer 31

1. TFIIIs bind internal promoter (downstream of +1 Inr) and upstream promoter
2. Poly III binds
3. Internal factors leave and transcription starts