HRR: Fundamentals of Gene Expression: Transcription I Flashcards
1
Q
Transcription Template
A
RNA synthesis is template directed.
The template strand of DNA
is always transcribed in 3´ to 5´ direction by RNA Polymerase
2
Q
Transcription Enzyme:
A
RNA Polymerase
3
Q
Enzymatic Reaction
A
(RNA)n + NTP —(Mg2+)–> (RNA)n+1 + PPi
NTP= ATP, CTP, UTP, GTP
4
Q
Processivity***
A
RNA Polymerases are processive enzymes that transcribe the template strand at rate of 50 nucleotides/sec.
5
Q
Main Steps of Transcription
A
- Termination
- Initiation
- Elongation
6
Q
do RNA have proofreading?
-why does this matter?
A
Polymerases do not have
3´ to 5´ exonuclease (proofreading) activity
7
Q
t/f both RNA and DNA synthesis, require a primer ed.
A
RNA polymerase initiates RNA synthesis de novo by joining 2 ribonucleotides together to form the first 3´to 5´ phosphodiester bond.
Unlike DNA synthesis, a primer is not required.
8
Q
Elongation Reaction of RNA Polymerases
A
Elongation Reaction of RNA Polymerases
9
Q
Elongation Reaction of RNA Polymerases
A
note
10
Q
t/f RNA has many different RNA polymerase
A
FALSE
RNA Polymerase: Prokaryotes have one type of RNA Polymerase
11
Q
Subunit Composition Number a b b´ s 2 1 1 1 Function Binds regulatory proteins with w subunit Phosphodiester bond formation, Grasps DNA Grasps DNA template Promoter recognition and initiation
A
Subunit Composition Number a b b´ s 2 1 1 1 Function Binds regulatory proteins with w subunit Phosphodiester bond formation, Grasps DNA Grasps DNA template Promoter recognition and initiation
12
Q
Gene Promoter: DNA sequence in the template strand of a gene that binds to RNA Polymerase.
In prokaryotes, the promoter of most genes has 2 consensus sequences as shown below.
A
. Gene Promoter: DNA sequence in the template strand of a gene that binds to RNA Polymerase.
In prokaryotes, the promoter of most genes has 2 consensus sequences as shown below.
13
Q
A consensus sequence is derived by
A
A consensus sequence is derived by determining the base found most frequently
at each position in the promoter region of the gene.
14
Q
- Initiation Step of Transcription
RNA Polymerase holoenzyme binds to DNA nonspecifically
A
- Initiation Step of Transcription
RNA Polymerase holoenzyme binds to DNA nonspecifically
15
Q
RNA Polymerase searches for ____ site
by “sliding” along double-stranded DNA.
-stops when :
A
RNA Polymerase searches for promoter site
by “sliding” along double-stranded DNA.
The s subunit binds SPECIFICALLY to the promoter
sequences to form the closed promoter complex
16
Q
closed promotor complex
A
bold
17
Q
RNA Polymerase unwinds 17 base pairs of DNA around
the initiation site to form the open promoter complex.
A
RNA Polymerase unwinds 17 base pairs of DNA around
the initiation site to form the open promoter complex.
18
Q
open promotor complex
A
the sigma subunit is released
19
Q
The first phosphodiester bond
is formed during initiation.
A
The first phosphodiester bond
is formed during initiation.
20
Q
unwound DNA
A
location
21
Q
_____ subunit is released upon
completion of the initiation step.
A
sigma subunit is released upon
completion of the initiation step.
22
Q
Elongation by core RNA Polymerase
A
Elongation by core RNA Polymerase
23
Q
. Initiation Step of Transcription
A
. Initiation Step of Transcription
RNA Polymerase Holoenzyme
• There is only one RNA Polymerase in prokaryotes. However, the RNA Polymerase
holoenzyme can initiate transcription of specific genes via the s subunit.
• Multiple s subunits exist, each one binds preferentially to a consensus sequence in the
promoter of a certain gene. Thus, specificity of the RNA Polymerase for a given promoter
is dependent on the s subunit that is part of the holoenzyme.
TTGACA TATAAT -35 -10 +1 s70 TNNCNCNCTTGAA CCCATNT \+1 s32 CTGGGNA TTGCA \+1 s54 Standard Promoter Promoter for heat shock genes Promoter for nitrogenstarvation genes Once the s subunit binds specifically to the consensus sequence, helicase activity in the RNA Polymerase holoenzyme unwinds 17 bp of DNA to form an open promoter complex.
24
Q
Open Promoter Complex
A
Once the s subunit binds specifically to the consensus sequence, helicase activity in the
RNA Polymerase holoenzyme unwinds 17 bp of DNA to form an open promoter complex.
25
Functions of the s Subunit
• RNA Polymerase holoenzyme is directed to the promoter due to high affinity binding
between the s subunit and the promoter sequence.
• The s subunit has much lower affinity for non-specific DNA sequences, which enables
RNA Polymerase holoenzyme to “slide” along the DNA and find promoter.
26
Elongation Step of Transcription
G-C base-pairing in newly synthesized
RNA forms hairpin, which disrupts its
association with DNA template strand
and halts the transcription reaction.
```
b) Rho-directed Termination: Rho protein
binds to specific sequences in newly
synthesized RNA. Rho protein has
ATP-dependent helicase activity that
dissociates RNA from template strand.
a) Template-directed Termination: Sequence in
DNA template strand of certain genes contain a
G-C rich repeat region followed by a region of A-T base pairs.
Transcription I [7]
Core Polymerase
Elongation
Core RNA Polymerase
The core RNA Polymerase generates
a transcription bubble that moves along
the chromosome. It consists of the
following:
• Unwound region = 17 bp of DNA
• RNA/DNA duplex = 12 bp
```
Elongation Step of Transcription
G-C base-pairing in newly synthesized
RNA forms hairpin, which disrupts its
association with DNA template strand
and halts the transcription reaction.
27
Transcription Bubble
```
The core RNA Polymerase generates
a transcription bubble that moves along
the chromosome. It consists of the
following:
• Unwound region = 17 bp of DNA
• RNA/DNA duplex = 12 bp
```
28
Termination Step of Transcription (prokaryotes)
a) Template-directed Termination: Sequence in
DNA template strand of certain genes contain a
G-C rich repeat region followed by a region of A-T base pairs.
```
b) Rho-directed Termination: Rho protein
binds to specific sequences in newly
synthesized RNA. Rho protein has
ATP-dependent helicase activity that
dissociates RNA from template strand.
```
Weak bonding between
A-U of RNA-DNA duplex
causes dissociation of RNA
from DNA template.
29
Weak bonding between
A-U of RNA-DNA duplex
causes dissociation of RNA
from DNA template.
Weak bonding between
A-U of RNA-DNA duplex
causes dissociation of RNA
from DNA template.
30
RNA Polymerase I
Types of RNA Polymerases
products: 45S Pre-ribosomal RNA [5.8S, 18S, 28S rRNA]
location: Nucleolus
31
RNA Polymerase II
RNA Polymerase II
RNA products: Pre-mRNAs, Primary miRNAs
snRNAs, lncRNAs
Location: Nucleus
32
RNA Polymerase III
products: tRNAs, 5S rRNA,
some snRNAs
location: Nucleus
33
Mitochondrial
RNA products: All mitochondrial RNAs
| location: Mitochondria
34
Basic Components of Eukaryotic Transcription by RNA Polymerases
Core RNA Polymerase: Transcription enzyme consisting of multiple protein subunits.
b) General Transcription Factors: Proteins required for basal transcription of all genes.
c) Specific Transcription Factors: Proteins that regulate transcription of specific target genes.
The composition and number of specific transcription factors that regulate each gene differ,
depending on its regulatory sequences. The following terminology is often used for specific
transcription factors:
• Activators (Transactivators) and Repressors (Transrepressors): Regulate transcription
by binding directly to DNA regulatory sequences in a gene. They can also bind to other
proteins including general transcription factors and mediator proteins.
• Mediator Proteins (Coactivators and Corepressors): Do not bind DNA directly, rather
they bind to and interact with general and/or specific transcription factors to regulate their
activity. Many mediator proteins function in modifying chromatin by epigenetic mechanisms.
35
Core RNA Polymerase: Transcription enzyme consisting of multiple protein subunits.
Core RNA Polymerase: Transcription enzyme consisting of multiple protein subunits.
b) General Transcription Factors: Proteins required for basal transcription of all genes.
c) Specific Transcription Factors: Proteins that regulate transcription of specific target genes.
The composition and number of specific transcription factors that regulate each gene differ,
depending on its regulatory sequences. The following terminology is often used for specific
transcription factors:
• Activators (Transactivators) and Repressors (Transrepressors): Regulate transcription
by binding directly to DNA regulatory sequences in a gene. They can also bind to other
proteins including general transcription factors and mediator proteins.
• Mediator Proteins (Coactivators and Corepressors): Do not bind DNA directly, rather
they bind to and interact with general and/or specific transcription factors to regulate their
activity. Many mediator proteins function in modifying chromatin by epigenetic mechanisms.
36
b) General Transcription Factors: Proteins required for basal transcription of all genes.
c) Specific Transcription Factors: Proteins that regulate transcription of specific target genes.
The composition and number of specific transcription factors that regulate each gene differ,
depending on its regulatory sequences. The following terminology is often used for specific
transcription factors:
• Activators (Transactivators) and Repressors (Transrepressors): Regulate transcription
by binding directly to DNA regulatory sequences in a gene. They can also bind to other
proteins including general transcription factors and mediator proteins.
• Mediator Proteins (Coactivators and Corepressors): Do not bind DNA directly, rather
they bind to and interact with general and/or specific transcription factors to regulate their
activity. Many mediator proteins function in modifying chromatin by epigenetic mechanisms.
37
c) Specific Transcription Factors: Proteins that regulate transcription of specific target genes.
The composition and number of specific transcription factors that regulate each gene differ,
depending on its regulatory sequences. The following terminology is often used for specific
transcription factors:
• Activators (Transactivators) and Repressors (Transrepressors): Regulate transcription
by binding directly to DNA regulatory sequences in a gene. They can also bind to other
proteins including general transcription factors and mediator proteins.
• Mediator Proteins (Coactivators and Corepressors): Do not bind DNA directly, rather
they bind to and interact with general and/or specific transcription factors to regulate their
activity. Many mediator proteins function in modifying chromatin by epigenetic mechanisms.
c) Specific Transcription Factors: Proteins that regulate transcription of specific target genes.
The composition and number of specific transcription factors that regulate each gene differ,
depending on its regulatory sequences. The following terminology is often used for specific
transcription factors:
• Activators (Transactivators) and Repressors (Transrepressors): Regulate transcription
by binding directly to DNA regulatory sequences in a gene. They can also bind to other
proteins including general transcription factors and mediator proteins.
• Mediator Proteins (Coactivators and Corepressors): Do not bind DNA directly, rather
they bind to and interact with general and/or specific transcription factors to regulate their
activity. Many mediator proteins function in modifying chromatin by epigenetic mechanisms.
38
Transcriptional Regulation: cis-Acting Elements and trans-Acting Factors
a) cis-Acting Elements: Regulatory DNA sequences specific to each gene, e.g., Core Promoter,
Proximal Promoter Elements, Enhancers, Silencers, Downstream Promoter Elements.
b) trans-Acting Factors: Regulatory proteins derived from genes other than the target gene,
e. g., General and Specific Transcription Factors (activators, repressors, mediator proteins)
39
Transcriptional Regulation
| cis-Acting Elements:
Regulatory DNA SEQUENCES specific to each gene, e.g., Core Promoter,
Proximal Promoter Elements, Enhancers, Silencers, Downstream Promoter Elements.
b) trans-Acting Factors: Regulatory proteins derived from genes other than the target gene,
e. g., General and Specific Transcription Factors (activators, repressors, mediator proteins)
40
Transcriptional Regulation:
trans-Acting Factors:
Regulatory PROTEINS derived from genes other than the target gene,
e.g., General and Specific Transcription Factors (activators, repressors, mediator proteins)
41
RNA Polymerase I: Enzyme that transcribes rRNA genes in the nucleolus.
(transcription in eukaryotes)
| RNA Polymerase I: Enzyme that transcribes rRNA genes in the nucleolus.
42
rRNA Genes
• The core promoter for rRNA genes is located:
• A human diploid cell contains approximately __#___ rRNA genes arranged in _____......
Each rRNA gene is separated by:
a) rRNA Genes
• The core promoter for rRNA genes is located in nontranscribed spacer DNA and spans the first 167 nt upstream of the transcription start site (+1).
• A human diploid cell contains approximately 400 rRNA genes arranged in tandem repeats
along the 5 acrocentric chromosomes [13, 14,15, 21, 22] in the nucleolus. Each rRNA gene is separated by nontranscribed spacer DNA
43
• RNA Polymerase I transcribes rRNA genes to generate 45S pre-rRNA. This precursor
transcript is processed into 18S, 28S and 5.8S rRNA.
• RNA Polymerase I transcribes rRNA genes to generate 45S pre-rRNA. This precursor
transcript is processed into 18S, 28S and 5.8S rRNA.
44
EM of nucleolar chromatin showing 3 tandem repeats of the rRNA gene separated
by nontranscribed spacer DNA. Each gene is transcribed by multiple RNA Polymerase I
complexes as indicated by the growing array of pre-rRNA transcripts
• Below: EM of nucleolar chromatin showing 3 tandem repeats of the rRNA gene separated
by nontranscribed spacer DNA. Each gene is transcribed by multiple RNA Polymerase I
complexes as indicated by the growing array of pre-rRNA transcripts
45
Components of RNA Polymerase I Transcription Complex
• Core RNA Polymerase I: Multimeric transcription enzyme consisting of 10-12 subunits
• Selectivity Factor 1 (SL1): Complex of 4 general transcription factors that binds to DNA
sequence in the core promoter of rRNA gene
• Upstream Binding Factor (UBF): Specific transcription factor that binds to DNA sequence
in the core promoter of rRNA gene and interacts with SL1 to promote assembly of a
transcriptionally-active RNA Polymerase I complex
46
RNA Polymerase I Complex
RNA Polymerase I Complex
47
• Core RNA Polymerase I: Multimeric transcription enzyme consisting of 10-12 subunits
• Core RNA Polymerase I: Multimeric transcription enzyme consisting of 10-12 subunits
• Selectivity Factor 1 (SL1): Complex of 4 general transcription factors that binds to DNA
sequence in the core promoter of rRNA gene
• Upstream Binding Factor (UBF): Specific transcription factor that binds to DNA sequence
in the core promoter of rRNA gene and interacts with SL1 to promote assembly of a
transcriptionally-active RNA Polymerase I complex
48
• Selectivity Factor 1 (SL1): Complex of 4 general transcription factors that binds to DNA
sequence in the core promoter of rRNA gene
• Selectivity Factor 1 (SL1): Complex of 4 general transcription factors that binds to DNA
sequence in the core promoter of rRNA gene
• Upstream Binding Factor (UBF): Specific transcription factor that binds to DNA sequence
in the core promoter of rRNA gene and interacts with SL1 to promote assembly of a
transcriptionally-active RNA Polymerase I complex
49
• Upstream Binding Factor (UBF): Specific transcription factor that binds to DNA sequence
in the core promoter of rRNA gene and interacts with SL1 to promote assembly of a
transcriptionally-active RNA Polymerase I complex
• Upstream Binding Factor (UBF): Specific transcription factor that binds to DNA sequence
in the core promoter of rRNA gene and interacts with SL1 to promote assembly of a
transcriptionally-active RNA Polymerase I complex
50
RNA Polymerase II: Enzyme that transcribes most genes to synthesize mRNA
RNA Polymerase II: Enzyme that transcribes most genes to synthesize mRNA
51
Core Promoter of Eukaryotic Genes
-def
The core promoter of most genes ranges between _____ bp in length and contains a combination of consensus sequence elements as shown below.
Genes vary greatly with respect to types of sequence
elements that are present in the core promoter.
Core Promoter of Eukaryotic Genes
The core promoter of a gene is defined as the minimal stretch of DNA sequence that is
sufficient to direct accurate initiation of transcription by RNA polymerase II. The core promoter
of most genes ranges between 60-120 bp in length and contains a combination of consensus
sequence elements as shown below. Genes vary greatly with respect to types of sequence
elements that are present in the core promoter.
52
• Initiator Sequence (Inr):
* TATA Box =
* TFIIB Recognition Element (BRE):
* Motif 10 Element (MTE):
* Downstream Promoter Element (DPE):
• Initiator Sequence (Inr): 6 bp sequence that spans the transcription start site (+1)
• TATA Box = TATA(A/T)A: Located -25 to -30 bp upstream of the transcription start site
• TFIIB Recognition Element (BRE): Approximately -35 bp upstream of transcription start site
• Motif 10 Element (MTE): Approximately +18 to +27 downstream of transcription start site
• Downstream Promoter Element (DPE): Approximately +28 to +32 downstream of the
transcription start site
53
Components of RNA Polymerase II Pre-initiation Complex
Transcription of all genes by RNA Polymerase II is dependent on assembly of the following
components into a pre-initiation complex on the core promoter of the gene. It also is called
the basal transcription complex.
* Core RNA Polymerase II:
* General Transcription Factors (TFs):
* Mediator:
The role of specific transcription
factors
Components of RNA Polymerase II Pre-initiation Complex
Transcription of all genes by RNA Polymerase II is dependent on assembly of the following
components into a pre-initiation complex on the core promoter of the gene. It also is called
the basal transcription complex.
• Core RNA Polymerase II: Multimeric enzyme consisting of 12 subunits. The C-terminal tail
domain (CTD) contains multiple Ser residues that are phosphorylated.
• General Transcription Factors (TFs): TFIID, TFIIA, TFIIB, TFIIE, TFIIF, TFIIH.
TFIID consists of: TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs).
• Mediator: Large coactivator complex that is required for the initiation step of transcription.
It consists of approximately 26 proteins that interact with the CTD of RNA Polymerase II,
general transcription factors and specific transcription factors.
Schematic of a pre-initiation complex
assembled on core promoter of a gene.
Specific transcription factors are not
shown. The role of specific transcription
factors is to promote assembly of this
complex on the core promoter of
specific genes to initiate transcription
by RNA Polymerase II above basal
levels.
54
RNA Polymerase II
C. TRANSCRIPTION IN EUKARYOTES
• Initiation of transcription in all genes is dependent on recruitment of RNA Polymerase II to the
the core promoter through assembly of a pre-initiation complex with general transcription
factors [TFIID, A, B, E, F, H] and the mediator complex.
55
Core promoter of any gene:
t/f Sequence elements in the
core promoter are the same among
individual genes.
false
Core promoter of any gene:
Sequence elements in the
core promoter varies among
individual genes.
56
• Current models hold that TFIID directs assembly of the pre-initiation complex, TFIID binds
first to the core promoter along with TFIIA. The TBP subunit binds directly to the TATA box if
present in the core promoter. However, most genes do not have a TATA box sequence in
the core promoter, thus, they are called “TATA-less”. As shown below, TAF subunits function
as a “molecular ruler” that positions TBP at the proper site in the core promoter.
• Current models hold that TFIID directs assembly of the pre-initiation complex, TFIID binds
first to the core promoter along with TFIIA. The TBP subunit binds directly to the TATA box if
present in the core promoter. However, most genes do not have a TATA box sequence in
the core promoter, thus, they are called “TATA-less”. As shown below, TAF subunits function
as a “molecular ruler” that positions TBP at the proper site in the core promoter.
57
Binding of TFII to core promoter:
what ensures that
TBP is positioned correctly relative to
the transcription start site?
```
Binding of TFII to core promoter:
Note that the TAF subunits form a
C-shaped bridge that facilitates
interactions between upstream and
downstream promoter elements such
as MTE and DPE. This ensures that
TBP is positioned correctly relative to
the transcription start site.
```
58
• TFIIB, TFIIF, TFIIE and TFIIH are recruited along with the core enzyme of RNA polymerase II.
This mechanism of sequential assembly ensures correct placement of RNA polymerase II
holoenzyme relative to the transcription start site.
• TFIIB, TFIIF, TFIIE and TFIIH are recruited along with the core enzyme of RNA polymerase II.
This mechanism of sequential assembly ensures correct placement of RNA polymerase II
holoenzyme relative to the transcription start site.
59
TFIIB:*
TFIIB:
Binds to BRE if present in the
core promoter.
60
```
TFIIF & TFIIE:
Assembly and stabilization of
initiation complex. You don’t
need to memorize functions
of TFIIF or TFIIE.
```
```
TFIIF & TFIIE:
Assembly and stabilization of
initiation complex. You don’t
need to memorize functions
of TFIIF or TFIIE.
```
61
TFIIH: *
-when activated it unwinds
TFIIH: Composed of multiple subunits for 2 main functions
1) Helicase activity- unwinding DNA strands in the core promoter
2) Protein kinase activity- phosphorylating CTD of RNA Polymerase II, which facilitates
promoter release for transcription of template DNA
62
Mediator: Protein complex required for initiation step by functioning as a link between
general and specific transcription factors. Mediator proteins are released from the initiation
complex upon phosphorylation of the CTD domain of RNA Polymerase II
Mediator: Protein complex required for initiation step by functioning as a link between
general and specific transcription factors. Mediator proteins are released from the initiation
complex upon phosphorylation of the CTD domain of RNA Polymerase II
63
) Elongation Step: Phosphorylation of CTD facilitates release of the RNA Polymerase II core
enzyme from the promoter. Additional elongation factors are recruited and activated
) Elongation Step: Phosphorylation of CTD facilitates release of the RNA Polymerase II core
enzyme from the promoter. Additional elongation factors are recruited and activated
64
Termination Step: Termination of transcription is coupled to synthesis of a polyadenylation
sequence in the 3´end of pre-mRNA. Cleavage and polyadenylation specificity factor (CPSF)
binds to this polyadenylation sequence, which results in downstream cleavage of the 3´-end.
An exonuclease is recruited that leads to dissociation of RNA Polymerase II from the template.
Termination Step: Termination of transcription is coupled to synthesis of a polyadenylation
sequence in the 3´end of pre-mRNA. Cleavage and polyadenylation specificity factor (CPSF)
binds to this polyadenylation sequence, which results in downstream cleavage of the 3´-end.
An exonuclease is recruited that leads to dissociation of RNA Polymerase II from the template.
65
Transcription overview
DNA (genes) --> RNA
66
RNA Polymerases are ____ enzymes that transcribe the template strand at rate of _____nucleotides/sec.
RNA Polymerases are processive enzymes that transcribe the template strand at rate of 50 nucleotides/sec.
67
A pharmaceutical Co develops an antibiotic that blocks transcription by specifically inhibiting activity of the sigma subunit of bacterial RNA Polymerase. Which of the following functions of RNA polymerase would be inhibited directly?
Recognition of consensus promoter sequence
68
Alice when walking in the woods eats some wild mushrooms =- the next day she vomits and has diarrhea. She goes to the hospital with liver failure and needs a transplant. It is found that the mushrooms contain a toxin that specifically inhibits RNA polymerase II.
Which of the following proces was most likely affected by the toxin?
Synthesis of pre-mRNA
| bc RNA polymerase II
69
REGULATION OF TRANSCRIPTION IN PROKARYOTES
1. lac Operon Model (Jacob & Monod)
A single polycistronic mRNA is generated that codes for all of the protein products.
note
70
1. lac Operon Model (Jacob & Monod)
An operon is a set of coordinately expressed genes.
A single polycistronic mRNA is generated that codes for all of the protein products.
lactose----(B-galactosidase)---> glucose + galactose
71
operon- def
An operon is a set of coordinately expressed genes.
72
Basic Components of Operon
a) Regulator Gene (i): Codes for repressor or activator protein
b) Operator (o): DNA regulatory sequence of the operon
c) Operon: Set of coordinately regulated target genes (z, y, a)
d) Inducer: Nutrient or metabolite such as lactose
Basic Components of Operon
a) Regulator Gene (i): Codes for repressor or activator protein b) Operator (o): DNA regulatory sequence of the operon
c) Operon: Set of coordinately regulated target genes (z, y, a) d) Inducer: Nutrient or metabolite such as lactose
73
lac Operon Model (Jacob & Monod)
- when remove B-galactosidase (in presence of lactose)
stop creating bacterial protein
74
T/F both DNA and RNA can be transcribed
false - only DNA!
75
T/F RNA is usually synthesized in 5´ to 3´ direction.
FALSE
| RNA is ALWAYS synthesized in 5´ to 3´ direction.
76
RNA grows in what direction?
5'-->3'
77
a core enzyme vs holoenzyme **
need a sigma subunit to make a holoenzyme
core enzyme : alpha2, W (omega i think), B and B'
holoeznyme: alpha2, W (omega i think), B - B' - sigma
78
B and B' subunits
form "pincers" that grasp the DNA template during transcription
79
core enzyme subunits
alpha2, W (omega i think), B and B'
80
(transcription in prokaryotes the initiation step)
Once the s subunit binds specifically to the consensus sequence, helicase activity in the
RNA Polymerase holoenzyme unwinds ____ bp of DNA to form an open promoter complex.
Once the s subunit binds specifically to the consensus sequence, helicase activity in the
RNA Polymerase holoenzyme unwinds 17 bp of DNA to form an open promoter complex.
81
**specificity of RNA Polymerase for a promoter is determined by the ___ subunit.
You don’t need to memorize these sequences or specific s subunits, rather, understand the
concept that specificity of RNA Polymerase for a promoter is determined by the s subunit.
82
transcription buble
• Unwound region =
• RNA/DNA duplex = bp
* Unwound region = 17 bp of DNA
| * RNA/DNA duplex = 12 bp
83
Role of Topoisomerases (in prokaryote transcription)
The transcription bubble causes overwinding of the DNA ahead of it. To prevent formation of positive supercoils, prokaryotes use gyrase (Type II topoisomerase) to create negative supercoils ahead of the bubble. As DNA rewinds, negative supercoils can form. Prokaryotes use Type I topoisomerases to relax negative supercoils behind the transcription bubble.
Role of Topoisomerases
The transcription bubble causes overwinding of the DNA ahead of it. To prevent formation of positive supercoils, prokaryotes use gyrase (Type II topoisomerase) to create negative supercoils ahead of the bubble. As DNA rewinds, negative supercoils can form. Prokaryotes use Type I topoisomerases to relax negative supercoils behind the transcription bubble.
84
```
b) Rho-directed Termination: Rho protein
binds to specific sequences in newly
synthesized RNA. Rho protein has
ATP-dependent helicase activity that
dissociates RNA from template strand.
```
```
b) Rho-directed Termination: Rho protein
binds to specific sequences in newly
synthesized RNA. Rho protein has
ATP-dependent helicase activity that
dissociates RNA from template strand.
```
85
which RNA polymerase take place in the nucleus?
RNA polymerase I and III
86
Core Promoter is an example of ____ element
cis
87
t/f there is only one promoter in a rRNA gene
FALSE
| there can be multiple
88
pre-mRNA only contains exons
false contains both introns and exons
| gets spliced to the mature mRNA which is only exons
89
t/f every gene has a TATA box
false- only about 15% do
90
TFIID **
TFIID consists of: TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs).
"directs"
91
lac operon
| Operator (o):
b) Operator (o): DNA regulatory sequence of the operon
92
Operon: **
Operon: Set of coordinately regulated target genes (z, y, a)
93
Absence of Lactose:
| lac operson
transcription is repressed
94
presence of lactose
lactose is an inducer
-protein is inactive
"de-press the gene"
95
when is the lac operon DEPRESSED
when lactose is present
because promotoer is accessible to RNA
96
low glucose and presence of lactose
result
97
Positive Regulation of lac Operon by cAMP
a) Presence of Lactose and Glucose: Transcription of the lac operon does not occur if glucose is still present. This is termed catabolite or glucose repression. Although the repressor
has been inactivated by addition of lactose, RNA Polymerase does not bind to the promoter. High glucose inhibits the expression of enzymes involved in breakdown of alternative sugars such as lactose by lowering cAMP.
Positive Regulation of lac Operon by cAMP
a) Presence of Lactose and Glucose: Transcription of the lac operon does not occur if glucose is still present. This is termed catabolite or glucose repression. Although the repressor
has been inactivated by addition of lactose, RNA Polymerase does not bind to the promoter. High glucose inhibits the expression of enzymes involved in breakdown of alternative sugars such as lactose by lowering cAMP.
98
t/f the core promoter can vary
TRUE
99
cis-Acting Regulatory Elements of a Gene
| Transcription II [5]
a) Core Promoter: The minimal stretch of DNA that is sufficient to direct accurate initiation of transcription by RNA polymerase II. It encompasses the transcription start site (+1) and ranges between 60-120 bp in length for most genes. Each core promoter contains a combin- ation of consensus DNA sequence elements such as Inr, TATA box, BRE, MTE and DPE.
b) Proximal Promoter Elements (PPE): cis-Acting sequences located -200 bp or closer to the transcription start site of the gene. They contain DNA sequences that are binding sites for specific transcription factors that function as either activators or repressors of basal transcription. The CCAAT box and GC box are PPEs found in many genes and are located frequently about 75 to 100 bp upstream of the transcription start site.
c) Enhancers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as activators of basal transcription. Enhancers regulate transcription independent of distance from the core promoter and can be located as far as 50 kilobases either upstream or downstream of the transcription start site. Enhancers can still activate transcription when polarity of DNA strands is flipped experimentally in the reverse orientation.
d) Silencers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as repressors of transcription. Like enhancers, silencers can function independent of distance from the core promoter and can be located many kilobases either upstream or downstream of the transcription start site.
100
***A single gene is regulated by multiple types of cis-acting elements.
A single gene is regulated by multiple types of cis-acting elements.
101
a) Core Promoter:
The minimal stretch of DNA that is sufficient to direct accurate initiation of transcription by RNA polymerase II. It encompasses the transcription start site (+1) and ranges between 60-120 bp in length for most genes. Each core promoter contains a combin- ation of consensus DNA sequence elements such as Inr, TATA box, BRE, MTE and DPE.
102
b) Proximal Promoter Elements (PPE): cis-Acting sequences located -200 bp or closer to the transcription start site of the gene. They contain DNA sequences that are binding sites for specific transcription factors that function as either activators or repressors of basal transcription. The CCAAT box and GC box are PPEs found in many genes and are located frequently about 75 to 100 bp upstream of the transcription start site.
c) Enhancers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as activators of basal transcription. Enhancers regulate transcription independent of distance from the core promoter and can be located as far as 50 kilobases either upstream or downstream of the transcription start site. Enhancers can still activate transcription when polarity of DNA strands is flipped experimentally in the reverse orientation.
d) Silencers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as repressors of transcription. Like enhancers, silencers can function independent of distance from the core promoter and can be located many kilobases either upstream or downstream of the transcription start site.
-upstream regulation very close to core promoter
103
c) Enhancers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as activators of basal transcription. Enhancers regulate transcription independent of distance from the core promoter and can be located as far as 50 kilobases either upstream or downstream of the transcription start site. Enhancers can still activate transcription when polarity of DNA strands is flipped experimentally in the reverse orientation.
- can be anywhere
| - independent of distance and orientation
104
d) Silencers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as repressors of transcription. Like enhancers, silencers can function independent of distance from the core promoter and can be located many kilobases either upstream or downstream of the transcription start site.
- independent of distance
d) Silencers: cis-Acting sequences that serve as DNA binding sites for specific transcription factors that function as repressors of transcription. Like enhancers, silencers can function independent of distance from the core promoter and can be located many kilobases either upstream or downstream of the transcription start site.
105
how many types of PPE
2
106
trans-Acting Factors: Proteins that are derived from a gene other than the target gene. trans- acting factors regulate transcription via cis-acting DNA sequences.
• Basal transcription of all genes is dependent on assembly of a pre-initiation complex on the
core promoter. In consists of RNA Polymerase II, general transcription factors and the mediator.
• Transcription of individual genes is regulated by specific transcription factors that function as activators to increase transcription of target genes above basal levels or as repressors to repress or silence transcription of target genes. Activators and repressors bind directly to DNA sequences in cis-acting elements, but they also have domains for recruiting other trans- acting factors that function in transcriptional regulation such as coactivators and corepressors.
trans-Acting Factors: Proteins that are derived from a gene other than the target gene. trans- acting factors regulate transcription via cis-acting DNA sequences.
• Basal transcription of all genes is dependent on assembly of a pre-initiation complex on the
core promoter. In consists of RNA Polymerase II, general transcription factors and the mediator.
• Transcription of individual genes is regulated by specific transcription factors that function as activators to increase transcription of target genes above basal levels or as repressors to repress or silence transcription of target genes. Activators and repressors bind directly to DNA sequences in cis-acting elements, but they also have domains for recruiting other trans- acting factors that function in transcriptional regulation such as coactivators and corepressors.
