rna structure and transcription
TRANSCRIPT
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RNA Structure a
a). Chemistry of RNA
i). Bases found in Rii). Ribose sugar
iii). RNA polynucleo
iv). Secondary and t
b). Characteristics of prok
i). Classes of prokaii). Structure of prok
c). Transcription initiation i
i). Transcription
ii). Promoter structu
iii). Prokaryotic RNiv). Initiation of tran
d). Regulation of the lacto
i). Function of the la
ii). Negative control:
iii). Positive control:
d
Transcription
A
ide chain
ertiary structure
ryotic RNA
yotic RNAaryotic messenger RNA
n prokaryotes
re
polymerase structurecription and the sigma cycle
e operon
ctose operon
Lac repressor and inducer
CAP and cAMP
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The major bases fou
DNA
Adenine
Cytosine
Guanine
Thymine
thymine-adenine base pair
d in DNA and RNA
RNA
Adenine
Cytosine
Guanine
Uracil (U)
uracil-adenine base pair
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Examples of modified
Dihydrouridine Pseudouridine 1-
1-methyladenosine 2-thiocytidine
b
ases found in RNA
ethylguanosine 7-methylguanosine
5-methylcytidine Ribothymine
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DNA polynucleotide chain
RNA polynucleotide chain
2· -OH makes
3·, 5· phosphodiester
bond unstable
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Secondary structure
Tertiary structure
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RNA p
closed promoter
open promoter
initiation
R
Transcription
lymerase
complex
omplex
elongation
termination
A product
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Promoter structure in pro
5¶
Promoter
-31-36
TTGACA
AACTGT
-30 region
84 79 53 45%82
T T G64
A C A 79
T
consensus seque
-30 -10
transcriptio
+1
[
aryotes
PuPuPuPuPuPuPuPu AUG
+1 +20-7-12
5¶mRNA
mRNA
TATAAT
ATATTA
-10 region
44
T96%
T95
A 59
A 51
A
ces
start site
Pribnow box
]
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Prokaryotic RNA polymera
RNA polymerase of bacteria i
Subunit Number
E 2
F(Rifampicin target) 1 F¶ 1
W 1
E FF¶W
holoenzyme core p
e structure
a multisubunit protein
Role
uncertain
forms phosphodiester bondsbinds DNA template
recognizes promoter and
facilitates initiation
FF¶ + W
olymerase sigma factor
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The function
the sigma subunit of RNA polyme
there are several different sigma f
specific for different sets of
sigma factor functions to ensu
stably to DNA only at pro
sigma destablizes nonspecific
sigma stabilizes specific bindi
this accelerates the search for
K
Any DNA
(nonspecific)
Core 2 X 1011
Holo 1 X 107
promoters vary in ³strength´ b
f sigma factor
rase is an ³initiation factor´
actors in E. coli that are
enes
e that RNA polymerase binds
oters
binding to non-promoter DNA
g to promoter DNA
promoter DNA
(M-1)
Promoter DNA
(specific)
1013 to 1015
~two orders of magnitude
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RNA pol
closed promote
the sigma subu
once initiation t
not need very h
sigma factor di
after a few elon
open promoter
the holoenzyme
promoter regio
W
sigma can re-bind
other core enzymes
W
Wmerase holoenzyme (+
Wfactor)
r complex (moderately stable)
it binds to the -10 region
kes place, RNA polymerase does
igh affinity for the promoter
sociates from the core polymerase
gation reactions
elongation takes place with
the core RNA polymerase
omplex (highly stable)
has very high affinity for
s because of sigma factor
The sigma cycle
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Mechanism of
RNA synthesis usually initiated wi
RNA chains are synthesized in a 5
A = T
U = A
RNA
NA synthesis
h ATP or GTP (the first nucleotide)
¶ to 3¶ direction
A = T
U = A
RNA
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lac I P O
promoter - operator
lac repressor
la
The lactose operon in E. col
F-galact
LACTOSE
F-galactosidase
the function of the lactose (lac) o
required to metabolize lactose for e
promoter binds CA
operator binds the l
Z lac Y lac A
i
sidase permease acetylase
GLUCOSE + GALACTOSE
eron is to produce the enzymes
ergy when it is required by the cell
and RNA polymerase
ac repressor
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Regulation of the lactose operon - neg
lac I P O
promoter - operator
lac repressor
lac I P la
the repressor tetra
RNA polymerase
la
RNA pol RNA polymerase is
tive control
Z lac Y lac A
er binds to the operator and prevents
from binding to the promoter
Z lac Y lac A
NO TRANSCRIPTION
blocked from the promoter
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when lactose becomes available, it i
allolactose (an intermediate in the h
one molecule of allolactose binds to
binding of allolactose results in a co
the conformational change results in
for the operator and dissociation of
Alleviation of negative control - action
allola
lac I P la
lac I P la
IPTG (isopropyl thiogalactoside)
is also used as a (non-physiological)
NO TRANSCRIPTION
taken up by the cell
drolysis of lactose) is produced
each of the repressor subunits
formational change in the repressor
decreased affinity of the repressor
he repressor from the DNA
f the inducer of the lac operon
tose
Z lac Y lac A
Z lac Y lac A
inducer
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lac I P la
RNA pol
O
repressor (with bound allolactose)
negative control (repression) is all
RNA polymerase cannot form a sta
Z lac Y lac A
NO TRANSCRIPTION
dissociates from the operator
viated, however...
ble complex with the promoter
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Affinity of lac repr
DNA Repressor
lac operator 2 X 1013
All other DNA 2 X 106
_________________________
Specificity1 107
_________________________
1 Specificity is the ratio of
(Ka for binding to operator DNA
ssor for DNA (M-1)
Repressor + Inducer
2 X 1010
2 X 106
_________________________
104
_________________________
) / (Ka for binding to random DNA)
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lac I P O la
Regulation of the lactose operon - posi
in the presence of both lactose and g
for the cell to metabolize lactose fo
in the absence of glucose and in theadvantageous to make use of the a
in the absence of glucose cells synth
cAMP1 serves as a positive regulator
cAMP binds the dimeric cAMP bindin
binding of cAMP increases the affinit
binding of CAP to the promoter facilit
1 cAMP = 3¶, 5¶ cyclic adenosine monophospha
active CAP
2 also termed catabolite activator protein
Z lac Y lac A
ive control
lucose it is not necessary
energy
resence of lactose it becomesailable lactose for energy
esize cyclic AMP (cAMP)
of catabolite operons (lac operon)
g protein (CAP)2
of CAP for the promoter
ates the binding of RNA polymerase
te
inactive CAPcAMP
+
NO TRANSCRIPTION
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lac I
lac repressor
la
F-galact
RNA pol
TRANSC
inactive repressor
Activation of lac operon transcription
the function of the lactose (lac)
required to metabolize lactose for e
Z lac Y lac A
sidase permease acetylase
RIPTION AND TRANSLATION OCCUR
peron is to produce the enzymes
nergy when it is required by the cell