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1
Q

Components in E coli RNAP

A

Five
Two alpha, one beta, one beta prime and one sigma70

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2
Q

Direction of RNA synthesis

A

5’ to 3’

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3
Q

What metal ions are needed in Ecoli RNAP?

A

Two Mg2+

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4
Q

How to identify promoter sequence in RNAP through experiment?

A
  • Add RNAP to radiolabelled DNA strand
  • Induce DNA cuts across the strand with DNase
  • lots of fragments formed of different sizes
  • conduct gel electrophoresis to determine binding site (without bands in sample with RNAP)
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5
Q

Location of initiation site in numbers

A

+1

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6
Q

Sequence of Pribnow box

A

TATAAT from 5’ to 3’

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7
Q

Terminology for Template strand

A

Non-coding strand or antisense

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8
Q

Terminology for non-template strand

A

Coding strand or sense

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9
Q

What binds to UP Elements?

A

C terminus of alpha subunit on RNAP

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10
Q

Where is Pribnow box located

A

-10

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11
Q

What binds to Pribnow box

A

Sigma subunit on RNAP thorugh recognition helix

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12
Q

Difference between genes with strong promoter and those with weak promoters

A

Strong: more transcription
Weak: lots of substitutions

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13
Q

Characteristics of UP Elements

A

AT rich

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14
Q

Components in promoter sequence of prokaryotes

A

-35 sequence
Pribnow box (-10)
UP Element

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15
Q

Which is the last step where transcription is reversible in prokaryotes

A

Complex formed between RNA polymerase and promoter

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16
Q

When is transcription in prokaryotes irreversible

A

Once DNA is unwounded in promoter region

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17
Q

What is holoenzyme in prokaryotic transcription

A

Sigma subunit plus core enzyme of RNAP

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18
Q

Structures important for nucleotide insertion in transcription of prokaryotes

A

Bridge helix and trigger loop

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19
Q

Intrinsic termination site in prokaryotic transcription

A
  1. GC rich inverted repeats —> forming a stem loop
  2. Non repeating segment between inverted repeats
  3. 6-8 U in transcript
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20
Q

Characteristics of Rho termination factor

A

Only in prokaryotes

ATP dependent helicase

Hexamer

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21
Q

How does Rho stop transcription in prokaryotes?

A

move along RNA transcript, unwind DNA:RNA hybrid and release DNA chain

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22
Q

Where does Rho bind to?

A

Rho utilization site (rut)
-C-rich

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23
Q

What happens when ribosome is on transcript of prokaryotes?

A

Rho factor cannot bind to transcript

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24
Q

How does rifampicin inhibits bacterial transcription?

A

binds to DNA RNA hybrid helix channel
—> blocks helix from forming

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25
How does actinomycin inhibit bacterial transcription?
act to intercalate within double helical DNA
26
How is 16S rRNA formed after transcription in prokaryotes?
cleaved by both RNase III and M16 to remove stem loop
27
How is 23S rRNA formed after prokaryotic transcription?
RNase III, M23
28
How is 5S rRNA formed after prokaryotic transcription?
M5 (only for cleaving 5S rRNA)
29
How is tRNA cleaved after prokaryotic transcription?
5’ of tRNA —> RNase P 3’ of tRNA —> RNase D
30
Function of RNase P
cleave 5’ of tRNA from primary transcript in prokaryotes
31
Function of RNase D
cleave 3’ of tRNA from primary transcript in prokaryotes
32
What happens after primary transcript formed for rRNA or tRNA in prokaryotes?
cleave spacer regions Process them (e.g. tRNA add CCA to end) Modification
33
Rank their steady state level from highest to lowest (tRNA, rRNA, mRNA)
rRNA>>tRNA>>mRNA
34
Effects of alpha amanitin to RNAP II
Strongly inhibited
35
A domain only found in RNAP II
C terminal domain with YSPTSPS repeats
36
Prokaryotic homolog of RPB4 in yeast RNAP II
sigma subunit —> promoter recognition
37
Prokaryotic homolog for RPB1
Beta prime
38
Feature of RPB 1 in RNAPII
YSPTSPS CTD
39
Feature of RPB4 in yeast RNAP II
Recognize promoter
40
In which steps of transcription does alpha amanitin act on?
1. After NTP inserted with open trigger loop —> prevent closure of loop 2.after intermediate formed in translocation steps —> prevent wedged trigger loop to open up
41
Promoters for RNAP II
1. TATA box 2. CAAT box (around -150 to -40) 3. GC box
42
Role of TFIID in transcription initiation
Has TBP component that recognizes TATA box —> then recruits TFIIA
43
Role of TFIIA in transcription initiation
stabilize TBP in TFIID after being recruited
44
Role of TFIIF in transcription initiation
bind to RNAP II —> bringing RNAP to promoter
45
TFIIH function in transcription initiation
Phosphorylate CTD in RNAP Unwind promoter with helicase activity
46
Two types of positive transcriptional regulation
1. Bind of ligand —> activator bind to transcript —> increase transcription efficiency 2. Bind of ligand —> dissociates activator from transcript —> inhibit transcription
47
Two types of negative transcriptional regulation
1. Ligand causes repressor to leave transcript —> allow transcription 2. Ligand causes repressor to bind transcript —> inhibit transcription
48
What does lac z code for?
beta galactosidase
49
what does lac y code for?
Gal permease
50
What does lac a code for?
Gal transacetylase
51
Function of beta galactosidase
i) breakdown lactose to galactose + glucose ii) change glycosidic linkage from 1-4 in lactose to 1-6 in allolactose (Side rxn)
52
Function of permease
Transporter of lactose
53
Function of Gal transacetylase
acetylates any unhydrolyzed lactose --> eliminate
54
What does lac i codes for?
repressor
55
Amino residue important for DNA adn Lac repressor interaction
Arginine, forming H bond with guanine
56
Function of allolactose
an inducer to repressor protein formed --> when bound, prevent repressor from binding to operator region
57
Function of repressor
form dimer --> bind to operator region to inhibit transcription
58
Structure of recognition helix in lac repressor
helix turn helix
59
How is catabolite activator protein (CAP) activated)?
low glucose level --> cAMP increase --> bind to N-term of CAP -->CAP activates
60
What happens after CAP is activated?
goes to binding region with AT rich inverted repeats (axis of symmetry)
61
What happens when there is low glucose levels + high lactose levels?
i) CAP activated (attached to cAMP) ii) lack lac repressor (as lactose is present) --> allolactose formed that bind to repressor -- increase transcription rate
62
What happens with High glucose + Low lactose?
i) CAP not activated (low amount of cAMP present) ii) lac repressor present (as lactose is absent) --> inhibit transcription
63
What happens with low levels of glucose and lactose?
i) CAP activated (high cAMP) ii) lac repressor present --> inhibit transcription
64
What happens with high levels of glucose and lactose?
i) CAP not activated (little cAMP) ii) lac repressor absent (allolactose bind to repressor) --> increase transcription
65
What does CAP bind to?
cAMP on N-term DNA on C-Term
66
How does CAP usually appear as?
Dimer
67
Three classes of DNA binding proteins
i) helix-turn-helix (HTH) motif ii) zinc-finger motif iii) Leucine zipper-basic region (bZIP)
68
Where does helix-turn helix (HTH) bind to DNA?
Major groove
69
What residues do zinc in zinc finger binds to?
Only Cys Half Cys, Half His
70
Characteristics of leucine zipper (bZIP)
i) DNA binding region (lot of basic aa like lysine and arginine) ii) Zipper region (Leu found in every 7 aa) --> Leu from both ends join tgt --> form coiled coil structure
71
How doese hormone receptor interact with DNA?
Zinc finger motif
72
What part of hormone receptor contributes most to conformation change during ligand binding?
helix 12
73
Effect of conformational change in hormone receptor to transcription
i) doesn't change receptor's affinity to DNA ii) allow recruitment of coactivator by increasing affinity to coactivator --> increase transcription
74
How does tamoxifen target hormone receptors?
act as antagonist of receptor --> helix 12 doesn't pack properly --> coactivator not recruited
75
Structure of hormone receptors
dimer

BCH311 (4 decks)

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