Molecular Biology
Molecular biology is usually described as that
domain of biology that seeks explanations in terms
of the structure and function of molecules of the
cell and sometimes of the whole organism,These
molecules are most frequently macromolecules
such as proteins,nucleic acids and carbohydrates,
or molecular assemblies such as lipidcontaining
membranes,and viruses.
Gene Expression
DNA
mRNA
Protein
Transcription
Translation
Transcription
Translation
34
3.4
20
M i n o r
G r o o ve
M aj o r
G r o o ve
GC
CG
AT
TA
CG
GC
AT
TA
TA
AT
GC
CG
GC
S t r an d s ar e
an t i p ar al l e l
Deoxyribonucleic acid (DNA)
is the genetic material
-Stores genetic information in
the form of a code,a linear
sequence of nucleotides
-- Replicated by copying the
strands using each as a
template for the production of
the complementary strand
D NA h a s a p h o s p h o d i e s t e r s u g a r b a c k b o n e,
Nucleosides (of DNA) – Precursors to Nucleotides
Nucleoside = base + sugar
Sugar = deoxyribose; 6 carbons,no OH on the 2nd (or 2’)
carbon; base is attached to carbon 1
The 4 Nucleotides of DNA
Ribose replaces deoxyribose; uracil replaces thymine
RNA
B e f o r e w e c o n ti n u e s o m e te r m i n o l o g y
N u c l e oti d e N am e T ab l e
P ur i ne s P yr i m i di ne s
A d e ni ne ( A ) G ua ni ne ( G ) C yt os i ne ( C ) T hym i n e ( T ) U r a c i l ( U )
N uc l e ot i de s i n D N A de ox ya d e ny l a t e de ox ygua nyl a t e de ox yc y t i dyl a t e de ox yt hym i d yl a t e
or t h ym i dyl a t e
N uc l e ot i de s i n R N A a d e ny l a t e gua nyl a t e c y t i dyl a t e ur i d yl a t e
A b b r e vi at i on s
N uc l e os i de
m onop hos pha t e s
A M P G M P C M P T M P U M P
N uc l e os i de
di p hos pha t e s
ADP G D P C D P T D P U D P
N uc l e os i de
t r i ph os p ha t e s
A T P G T P C T P TTP U T P
F or de o x y nuc l e o t i de s a dd ' d' i n f r ont o f t he a b ove t hr e e,
e.g.,AMP is a ribonucleotide,dAMP is a deoxyribonucleotide
In DNA and RNA,
nucleotides are held
together by
phosphodiester
bonds.
Classes of RNA
Class Function Position
hnRNA 成熟 mRNA的前体 核内
snRNA 参与 hnRNA的剪接、转运 核内
scRNA 蛋白质内质网定位合成的信 号识别体的组成成分 胞内
rRNA 核蛋白体组成成分
tRNA 转运氨基酸
mRNA 蛋白质合成模板
Stem loopStems are double-stranded
regions of RNA that are
A-form helices,They follow
Watson-Crick base pairing
rules,
(DNA is typically a B-form
helix).
Stem-loops are common elements of RNA structure
Hierarchy of RNA folding
RNA
secondary
structure
motifs
Secondary structure diagram Tertiary structure diagram
Cr.LSU rRNA intron Tetrahymena rRNA intron
1,Key Terms in Transcription
Coding strand(Sense strand) of DNA has the same sequence as
mRNA.
The antisense strand (Template strand) of DNA is
complementary to the sense strand,and is the one that acts as the
template for synthesis of mRNA.
RNA polymerases are enzymes that synthesize RNA using a
DNA template (formally described as DNA-dependent RNA
polymerases).
Promoter is a region of DNA involved in binding of RNA
polymerase to initiate transcription.
Startpoint (Startsite) refers to the position on DNA corresponding
to the first base incorporated into RNA.
Terminator is a sequence of DNA,represented at the end of the
transcript,that causes RNA polymerase to terminate transcription.
Key Terms in Transcription
Transcription unit is the distance between sites of initiation
and termination by RNA polymerase; may include more than
one gene.
Downstream identifies sequences proceeding farther in the
direction of expression; for example,the coding region is
downstream of the initiation codon.
Upstream identifies sequences proceeding in the opposite
direction from expression; for example,the bacterial promoter
is upstream from the transcription unit,the initiation codon is
upstream of the coding region.
Primary transcript is the original unmodified RNA product
corresponding to a transcription unit.
Transcription,similar to replication
but different
1,Polymerases use ribonucleotides (NTPs)
and Uracil instead of Thymine
2,Produces mainly single-stranded molecules
that are complementary to one strand of the
DNA template (or non-coding strand)
3,Only part of the genome is transcribed –
“functional genome” or,transcriptome”
2,Transcription,Basic Enzymology
1,DNA-dependent RNA Polymerases
2,RNA Pols polymerize in the 5’ to 3’ direction
(rNTP added only to the 3’ end)
3,3’ OH of chain reacts with the a PO4 of
incoming rNTP,liberating pyrophosphate
4,Added ribonucleotide follows Watson-Crick
pairing rules,determined by template strand
5,RNA polymerases don’t need a primer,but do
need DS DNA
E,coli RNA polymerases (RNAPs)
а- 2 copies
b - 1 copy
b’ - 1 copy
s - 1 copy
Core
holoenzyme
There are multiple forms of s.
Subunit complexity of RNA polymerases
3-D images of core and
holoenzyme
Core Holoenzyme
Holoenzyme for RNA
polymerase from E,coli
S i m i l a r t o D N A p o l y m e r a s e I K l e n o w
C h a n n e l w i t h p r o b a b l e D N A - b i n d i n g
a n d a c t i v e s i t e s,2 5 A n g s t r o m s i n
d i a m e t e r a n d 5 5 A n g s t r o m s l o n g
( e n o u g h f o r a b o u t 1 6 b p o f D N A ),
V i e w p e r p e n d i c u l a r t o c h a n n e l,
"t h u m b "
"f i n g e r s " D a r s t,S,A,,E,W,K u b a l e k & R,D,
K o r n b e r g ( 1 9 8 9 ) N a t u r e 3 4 0,7 3 0 - 7 3 2,
( vi e w f l i p p e d 1 8 0 )
o
A
o
1 0 0 t h i c k
1 6 0 A
o
1 0 0 A
o
H o l o e n z y m e f o r R N A p o l y m e r a s e f r o m E,c o l i a b b 's2 2 7 r e s o l u t i o nA
o
3,The transcription reaction has
three stages
Initiation describes the stages of transcription up to
synthesis of the first bond in RNA,This includes binding of
RNA polymerase to the promoter and melting a short
region of DNA into single strands.
Elongation is the stage in a macromolecular synthesis
reaction (replication,transcription,or translation) when the
nucleotide or polypeptide chain is being extended by the
addition of individual subunits.
Termination is a separate reaction that ends a
macromolecular synthesis reaction (replication,
transcription,or translation),by stopping the addition of
subunits,and (typically) causing disassembly of the
synthetic apparatus.
Template recognition
Template recognition begins with the binding of
RNA polymerase to the double-stranded DNA at a
promoter to form a "closed complex",Then the
strands of DNA are separated to form the "open
complex" that makes the template strand available
for base pairing with ribonucleotides,The
transcription bubble is created by a local
unwinding that begins at the site bound by RNA
polymerase.
Binding
Occurs at promoters
(-10,-35) or σ70 most common type
Recognized by σ70 subunit
E,coli has at least 2 other promoter
types
– Recognized by other s factors
Sigma (s) subunit recognizes promoter sequences
a2bb’w core cannot bind at promoter without s = (a2bb’ws)
– s decreases affinity for non-specific regions by ~104
– RNAP diffuses along dsDNA rapidly looking for promoter in a
1-D random walk
Rate constant for promoter binding is 1010 M-1! >100x a 3D
binding mechanism
No need to unwind DNA double helix
there are several types of s factors in E,coli used for different
scenarios,
– e.g,s70,the most common one
– e.g,s32 for heat shock response
s32 recognizes different promoter from the right
Consensus,TTGACA 17+/- 1bp TATAAT
-35 -10
Typical E,coli promoter structure
UP = upstream promoter (seen in highly expressed genes)
changes to promoter sequences and/or spacing of the elements
dramatically alters the rate of initiation (control)
a s70 s70
The very strong rRNA promoter has an
extra upstream element,UP.
Recognized by the?subunit,
strengthens binding of Polymerase to the
promoter,Fig,6.11
Initiation
Polymerase bound
tightly to promoter,
“Closed Complex”
Polymerase unwinds
DNA (-10 to +3),
“Open Complex”
Starts synthesizing
RNA
Fig,6.7
Events at
initiation
of
transcriptio
n
Sites on RNA polymerase core
Enzyme covers about 60 bp of DNA,with about
17 bp unwound = transcription bubble.
The bubble must contact the active site for
polymerization.
At the beginning of the bubble,the DNA is
unwound,implicating a helicase activity.
At the end of the bubble,the DNA is rewound.
Elongation
A,After ~10 nucleotides have been added,5’ end ribonucleotide
unpairs from template.
B,The s subunit dissociates from core.
C,The size of RNA-DNA hybrid maintained during elongation.
D,Sigma recycles to new polymerase molecules.
Termination
Two types of termination events in E,coli
– Rho independent
– Rho dependent
Rho independent also called intrinsic
– Triggered by an Inverted Repeat (IR) that
occurs downstream of Rho independent
genes
– IR followed by an A-rich region
IR in DNA produces a stem-loop in RNA.
Stem-loop
formation
competes with
the RNA-DNA
hybrid (Open
Complex).
Causing DNA
helix to reform
(Closed
complex).
Rho-dependent termination
Rho is a RNA helicase,and an ATPase
Travels along RNA chasing polymerase
When polymerase stalls at an IR (no A-T rich
region),Rho dissociates the RNA-DNA hybrid
DNA helix closes
Fig,6.50
Rho in action
Rho binds
transcripts at
stretches of ~100
nt free of 2nd
structure and rich
in cytosines.
Rho is a hexamer
helicase,like DnaB
except prefers RNA.
Can unwind RNA-DNA
hybrids.
Transcription cycle
Initiation
– Holoenzyme binds to the promoter,unwinds DNA,
and forms phosphodiester bonds between 7 to 12
nucleotides
– Need s
Elongation
– s dissociates
– Core elongates RNA with high processivity
Termination
– Polymerase dissociates from template DNA and
releases new RNA
– Often use r.
Transcription Cycle
H ow do e s s i g ma ass o ci a t e w i t h a p r o m o te r?
T h e s s u b u n it a p p e ar s t o h a v e t w o s eg m en t s t ha t c o n ta c t t he b a s e s o f D N A m o l e c u l e
t h r o u g h t he m a j or g r o o v e,I t d o e s t h is w h il e i t i s as s oc i a t ed w it h t he co r e e n z y m e.
-10-35
4,Role of Sigma Factor
Looking up from below the DNA.
Mode of action of s factors
The s factor causes RNA polymerase to be
selective in its choice of initiation sites by
affecting the dissociation rate of polymerase from
DNA.
– Core dissociates from general DNA with a half-time of
60 min; use in elongation.
– Holoenzyme dissociates from general DNA with a half-
time of 1 sec!
– Holoenzyme dissociates from promoter DNA with a
half-time of hours (in absence of rNTPs),
1 2 3 4
2,1 2,2 2,3 2,4
C
o
re
B
i
n
d
i
n
g
D
N
A
M
e
l
t
i
n
g
-
10
R
e
c
o
g
n
i
t
i
o
n
A
c
t
i
v
at
o
r
C
o
n
t
ac
t
-
35
R
e
c
o
g
n
i
t
i
o
n
4,1 4,2
H e l i x - T u r n - H e l i x
2.1 - Core binding
2.3 - DNA melting
2.4 - (-10) recognition
4.1 - Activators bind
4.2 - (-35) recognition
Conserved regions of sigma factors
Pol Core has non-specific affinity for DNA
that is reduced by s.
Sliding and/or Hopping
Promoter binding by RNA Pol too fast for random
collision (diffusion) theory
Facilitated diffusion theory (2 models)
– Sliding (not much evidence for it)
– Hopping,random association/dissociation
Could be used on supercoiled,compact
molecules
RNA polymerase -
Hopping about
5,E,coli RNA polymerase
Functions of other subunits:
1,a - binds the UP element found upstream of
very strong promoters (rRNA),and activators
2,b - active site of Pol,also binds nascent RNA,
RNA-DNA hybrid,and DS DNA in front of the
bubble
3,b’ – also binds nascent RNA,RNA-DNA hybrid,
and DS DNA in front of the bubble
Role of a subunit in assembly of RNA
polymerase and other functions
Drugs that inhibit E,coli RNAP
Rifampicin (利福平） - blocks at initiation
(Open Complex forms),rifampicin
resistance mutations mapped to the b
subunit.
Streptolydigin（利迪链霉素） - inhibits
elongation stage,resistance mutations also
map to b subunit.
What do initiation and elongation have in
common? Phosphodiester bond formation,
Suggests β subunit is key.
Direct evidence for β
active site by,Affinity
Labeling”.
(1) Reagent in (a) resembles
ATP,but it reacts with NH2
groups when binds to active
site.
(2) Then add radioactive α -
32P-UTP,which is added (i.e.,
polymerized) onto reagent I.
Fig,6.33
(3) Separate enzyme
subunits by SDS-
polyacrylamide gel
electrophoresis,and see
which ones are radioactive,
Only the β subunit is
highly radioactive.
β subunit must have active
site.
Fig,6.34
Nudler et al.,1998,Science 281,pg,425
Fig,6.39
Thermus aquaticus RNAP core.,The Claw”
Backsliding and Editing
1,If wrong nucleotide incorporated,elongation
can become arrested.
2,Backsliding now competes with elongation:
– Pol backs up,extruding some of nascent RNA
3,Gre proteins activate RNAP core to cleave
small piece that has wrong nucleotide,
4,Pol starts elongatin gagain,
Square is the next NTP
to be added.
Green – nascent RNA
that will be cleaved off
Red –,older RNA”
RNAP core
Summary
Some terms about
transcription
Prokaryotic
– Apparatus
– Mechanism
– Regulation
Thanks!
Any Question?
Molecular biology is usually described as that
domain of biology that seeks explanations in terms
of the structure and function of molecules of the
cell and sometimes of the whole organism,These
molecules are most frequently macromolecules
such as proteins,nucleic acids and carbohydrates,
or molecular assemblies such as lipidcontaining
membranes,and viruses.
Gene Expression
DNA
mRNA
Protein
Transcription
Translation
Transcription
Translation
34
3.4
20
M i n o r
G r o o ve
M aj o r
G r o o ve
GC
CG
AT
TA
CG
GC
AT
TA
TA
AT
GC
CG
GC
S t r an d s ar e
an t i p ar al l e l
Deoxyribonucleic acid (DNA)
is the genetic material
-Stores genetic information in
the form of a code,a linear
sequence of nucleotides
-- Replicated by copying the
strands using each as a
template for the production of
the complementary strand
D NA h a s a p h o s p h o d i e s t e r s u g a r b a c k b o n e,
Nucleosides (of DNA) – Precursors to Nucleotides
Nucleoside = base + sugar
Sugar = deoxyribose; 6 carbons,no OH on the 2nd (or 2’)
carbon; base is attached to carbon 1
The 4 Nucleotides of DNA
Ribose replaces deoxyribose; uracil replaces thymine
RNA
B e f o r e w e c o n ti n u e s o m e te r m i n o l o g y
N u c l e oti d e N am e T ab l e
P ur i ne s P yr i m i di ne s
A d e ni ne ( A ) G ua ni ne ( G ) C yt os i ne ( C ) T hym i n e ( T ) U r a c i l ( U )
N uc l e ot i de s i n D N A de ox ya d e ny l a t e de ox ygua nyl a t e de ox yc y t i dyl a t e de ox yt hym i d yl a t e
or t h ym i dyl a t e
N uc l e ot i de s i n R N A a d e ny l a t e gua nyl a t e c y t i dyl a t e ur i d yl a t e
A b b r e vi at i on s
N uc l e os i de
m onop hos pha t e s
A M P G M P C M P T M P U M P
N uc l e os i de
di p hos pha t e s
ADP G D P C D P T D P U D P
N uc l e os i de
t r i ph os p ha t e s
A T P G T P C T P TTP U T P
F or de o x y nuc l e o t i de s a dd ' d' i n f r ont o f t he a b ove t hr e e,
e.g.,AMP is a ribonucleotide,dAMP is a deoxyribonucleotide
In DNA and RNA,
nucleotides are held
together by
phosphodiester
bonds.
Classes of RNA
Class Function Position
hnRNA 成熟 mRNA的前体 核内
snRNA 参与 hnRNA的剪接、转运 核内
scRNA 蛋白质内质网定位合成的信 号识别体的组成成分 胞内
rRNA 核蛋白体组成成分
tRNA 转运氨基酸
mRNA 蛋白质合成模板
Stem loopStems are double-stranded
regions of RNA that are
A-form helices,They follow
Watson-Crick base pairing
rules,
(DNA is typically a B-form
helix).
Stem-loops are common elements of RNA structure
Hierarchy of RNA folding
RNA
secondary
structure
motifs
Secondary structure diagram Tertiary structure diagram
Cr.LSU rRNA intron Tetrahymena rRNA intron
1,Key Terms in Transcription
Coding strand(Sense strand) of DNA has the same sequence as
mRNA.
The antisense strand (Template strand) of DNA is
complementary to the sense strand,and is the one that acts as the
template for synthesis of mRNA.
RNA polymerases are enzymes that synthesize RNA using a
DNA template (formally described as DNA-dependent RNA
polymerases).
Promoter is a region of DNA involved in binding of RNA
polymerase to initiate transcription.
Startpoint (Startsite) refers to the position on DNA corresponding
to the first base incorporated into RNA.
Terminator is a sequence of DNA,represented at the end of the
transcript,that causes RNA polymerase to terminate transcription.
Key Terms in Transcription
Transcription unit is the distance between sites of initiation
and termination by RNA polymerase; may include more than
one gene.
Downstream identifies sequences proceeding farther in the
direction of expression; for example,the coding region is
downstream of the initiation codon.
Upstream identifies sequences proceeding in the opposite
direction from expression; for example,the bacterial promoter
is upstream from the transcription unit,the initiation codon is
upstream of the coding region.
Primary transcript is the original unmodified RNA product
corresponding to a transcription unit.
Transcription,similar to replication
but different
1,Polymerases use ribonucleotides (NTPs)
and Uracil instead of Thymine
2,Produces mainly single-stranded molecules
that are complementary to one strand of the
DNA template (or non-coding strand)
3,Only part of the genome is transcribed –
“functional genome” or,transcriptome”
2,Transcription,Basic Enzymology
1,DNA-dependent RNA Polymerases
2,RNA Pols polymerize in the 5’ to 3’ direction
(rNTP added only to the 3’ end)
3,3’ OH of chain reacts with the a PO4 of
incoming rNTP,liberating pyrophosphate
4,Added ribonucleotide follows Watson-Crick
pairing rules,determined by template strand
5,RNA polymerases don’t need a primer,but do
need DS DNA
E,coli RNA polymerases (RNAPs)
а- 2 copies
b - 1 copy
b’ - 1 copy
s - 1 copy
Core
holoenzyme
There are multiple forms of s.
Subunit complexity of RNA polymerases
3-D images of core and
holoenzyme
Core Holoenzyme
Holoenzyme for RNA
polymerase from E,coli
S i m i l a r t o D N A p o l y m e r a s e I K l e n o w
C h a n n e l w i t h p r o b a b l e D N A - b i n d i n g
a n d a c t i v e s i t e s,2 5 A n g s t r o m s i n
d i a m e t e r a n d 5 5 A n g s t r o m s l o n g
( e n o u g h f o r a b o u t 1 6 b p o f D N A ),
V i e w p e r p e n d i c u l a r t o c h a n n e l,
"t h u m b "
"f i n g e r s " D a r s t,S,A,,E,W,K u b a l e k & R,D,
K o r n b e r g ( 1 9 8 9 ) N a t u r e 3 4 0,7 3 0 - 7 3 2,
( vi e w f l i p p e d 1 8 0 )
o
A
o
1 0 0 t h i c k
1 6 0 A
o
1 0 0 A
o
H o l o e n z y m e f o r R N A p o l y m e r a s e f r o m E,c o l i a b b 's2 2 7 r e s o l u t i o nA
o
3,The transcription reaction has
three stages
Initiation describes the stages of transcription up to
synthesis of the first bond in RNA,This includes binding of
RNA polymerase to the promoter and melting a short
region of DNA into single strands.
Elongation is the stage in a macromolecular synthesis
reaction (replication,transcription,or translation) when the
nucleotide or polypeptide chain is being extended by the
addition of individual subunits.
Termination is a separate reaction that ends a
macromolecular synthesis reaction (replication,
transcription,or translation),by stopping the addition of
subunits,and (typically) causing disassembly of the
synthetic apparatus.
Template recognition
Template recognition begins with the binding of
RNA polymerase to the double-stranded DNA at a
promoter to form a "closed complex",Then the
strands of DNA are separated to form the "open
complex" that makes the template strand available
for base pairing with ribonucleotides,The
transcription bubble is created by a local
unwinding that begins at the site bound by RNA
polymerase.
Binding
Occurs at promoters
(-10,-35) or σ70 most common type
Recognized by σ70 subunit
E,coli has at least 2 other promoter
types
– Recognized by other s factors
Sigma (s) subunit recognizes promoter sequences
a2bb’w core cannot bind at promoter without s = (a2bb’ws)
– s decreases affinity for non-specific regions by ~104
– RNAP diffuses along dsDNA rapidly looking for promoter in a
1-D random walk
Rate constant for promoter binding is 1010 M-1! >100x a 3D
binding mechanism
No need to unwind DNA double helix
there are several types of s factors in E,coli used for different
scenarios,
– e.g,s70,the most common one
– e.g,s32 for heat shock response
s32 recognizes different promoter from the right
Consensus,TTGACA 17+/- 1bp TATAAT
-35 -10
Typical E,coli promoter structure
UP = upstream promoter (seen in highly expressed genes)
changes to promoter sequences and/or spacing of the elements
dramatically alters the rate of initiation (control)
a s70 s70
The very strong rRNA promoter has an
extra upstream element,UP.
Recognized by the?subunit,
strengthens binding of Polymerase to the
promoter,Fig,6.11
Initiation
Polymerase bound
tightly to promoter,
“Closed Complex”
Polymerase unwinds
DNA (-10 to +3),
“Open Complex”
Starts synthesizing
RNA
Fig,6.7
Events at
initiation
of
transcriptio
n
Sites on RNA polymerase core
Enzyme covers about 60 bp of DNA,with about
17 bp unwound = transcription bubble.
The bubble must contact the active site for
polymerization.
At the beginning of the bubble,the DNA is
unwound,implicating a helicase activity.
At the end of the bubble,the DNA is rewound.
Elongation
A,After ~10 nucleotides have been added,5’ end ribonucleotide
unpairs from template.
B,The s subunit dissociates from core.
C,The size of RNA-DNA hybrid maintained during elongation.
D,Sigma recycles to new polymerase molecules.
Termination
Two types of termination events in E,coli
– Rho independent
– Rho dependent
Rho independent also called intrinsic
– Triggered by an Inverted Repeat (IR) that
occurs downstream of Rho independent
genes
– IR followed by an A-rich region
IR in DNA produces a stem-loop in RNA.
Stem-loop
formation
competes with
the RNA-DNA
hybrid (Open
Complex).
Causing DNA
helix to reform
(Closed
complex).
Rho-dependent termination
Rho is a RNA helicase,and an ATPase
Travels along RNA chasing polymerase
When polymerase stalls at an IR (no A-T rich
region),Rho dissociates the RNA-DNA hybrid
DNA helix closes
Fig,6.50
Rho in action
Rho binds
transcripts at
stretches of ~100
nt free of 2nd
structure and rich
in cytosines.
Rho is a hexamer
helicase,like DnaB
except prefers RNA.
Can unwind RNA-DNA
hybrids.
Transcription cycle
Initiation
– Holoenzyme binds to the promoter,unwinds DNA,
and forms phosphodiester bonds between 7 to 12
nucleotides
– Need s
Elongation
– s dissociates
– Core elongates RNA with high processivity
Termination
– Polymerase dissociates from template DNA and
releases new RNA
– Often use r.
Transcription Cycle
H ow do e s s i g ma ass o ci a t e w i t h a p r o m o te r?
T h e s s u b u n it a p p e ar s t o h a v e t w o s eg m en t s t ha t c o n ta c t t he b a s e s o f D N A m o l e c u l e
t h r o u g h t he m a j or g r o o v e,I t d o e s t h is w h il e i t i s as s oc i a t ed w it h t he co r e e n z y m e.
-10-35
4,Role of Sigma Factor
Looking up from below the DNA.
Mode of action of s factors
The s factor causes RNA polymerase to be
selective in its choice of initiation sites by
affecting the dissociation rate of polymerase from
DNA.
– Core dissociates from general DNA with a half-time of
60 min; use in elongation.
– Holoenzyme dissociates from general DNA with a half-
time of 1 sec!
– Holoenzyme dissociates from promoter DNA with a
half-time of hours (in absence of rNTPs),
1 2 3 4
2,1 2,2 2,3 2,4
C
o
re
B
i
n
d
i
n
g
D
N
A
M
e
l
t
i
n
g
-
10
R
e
c
o
g
n
i
t
i
o
n
A
c
t
i
v
at
o
r
C
o
n
t
ac
t
-
35
R
e
c
o
g
n
i
t
i
o
n
4,1 4,2
H e l i x - T u r n - H e l i x
2.1 - Core binding
2.3 - DNA melting
2.4 - (-10) recognition
4.1 - Activators bind
4.2 - (-35) recognition
Conserved regions of sigma factors
Pol Core has non-specific affinity for DNA
that is reduced by s.
Sliding and/or Hopping
Promoter binding by RNA Pol too fast for random
collision (diffusion) theory
Facilitated diffusion theory (2 models)
– Sliding (not much evidence for it)
– Hopping,random association/dissociation
Could be used on supercoiled,compact
molecules
RNA polymerase -
Hopping about
5,E,coli RNA polymerase
Functions of other subunits:
1,a - binds the UP element found upstream of
very strong promoters (rRNA),and activators
2,b - active site of Pol,also binds nascent RNA,
RNA-DNA hybrid,and DS DNA in front of the
bubble
3,b’ – also binds nascent RNA,RNA-DNA hybrid,
and DS DNA in front of the bubble
Role of a subunit in assembly of RNA
polymerase and other functions
Drugs that inhibit E,coli RNAP
Rifampicin (利福平） - blocks at initiation
(Open Complex forms),rifampicin
resistance mutations mapped to the b
subunit.
Streptolydigin（利迪链霉素） - inhibits
elongation stage,resistance mutations also
map to b subunit.
What do initiation and elongation have in
common? Phosphodiester bond formation,
Suggests β subunit is key.
Direct evidence for β
active site by,Affinity
Labeling”.
(1) Reagent in (a) resembles
ATP,but it reacts with NH2
groups when binds to active
site.
(2) Then add radioactive α -
32P-UTP,which is added (i.e.,
polymerized) onto reagent I.
Fig,6.33
(3) Separate enzyme
subunits by SDS-
polyacrylamide gel
electrophoresis,and see
which ones are radioactive,
Only the β subunit is
highly radioactive.
β subunit must have active
site.
Fig,6.34
Nudler et al.,1998,Science 281,pg,425
Fig,6.39
Thermus aquaticus RNAP core.,The Claw”
Backsliding and Editing
1,If wrong nucleotide incorporated,elongation
can become arrested.
2,Backsliding now competes with elongation:
– Pol backs up,extruding some of nascent RNA
3,Gre proteins activate RNAP core to cleave
small piece that has wrong nucleotide,
4,Pol starts elongatin gagain,
Square is the next NTP
to be added.
Green – nascent RNA
that will be cleaved off
Red –,older RNA”
RNAP core
Summary
Some terms about
transcription
Prokaryotic
– Apparatus
– Mechanism
– Regulation
Thanks!
Any Question?
