4. 4
HOW RNA POLYMERASE FINDHOW RNA POLYMERASE FIND
CORRECT SITECORRECT SITE
E.coli 4X10E.coli 4X1033
Transcription sites in 4+106Transcription sites in 4+106
bp long DNA.bp long DNA.
Humans 105 transcription sites in 3X10Humans 105 transcription sites in 3X1099
bp of DNA.bp of DNA.
How efficiently the entire genome isHow efficiently the entire genome is
scanned for the right gene unit for the startscanned for the right gene unit for the start
of transcription.of transcription.
8. 8
The synthesis of RNA molecules usingThe synthesis of RNA molecules using
DNA strands as the templates so that theDNA strands as the templates so that the
genetic information can be transferredgenetic information can be transferred
from DNA to RNA.from DNA to RNA.
Transcription
9. 9
ObjectivesObjectives
Understand the structure of RNAUnderstand the structure of RNA
polymerasespolymerases
Understand the phases of the transcriptionUnderstand the phases of the transcription
cyclecycle
Understand the differences betweenUnderstand the differences between
transcription and replicationtranscription and replication
10. 10
Both processes use DNA as theBoth processes use DNA as the
template.template.
Phosphodiester bonds are formed inPhosphodiester bonds are formed in
both cases.both cases.
Both synthesis directions are from 5´Both synthesis directions are from 5´
to 3´.to 3´.
Similarity betweenSimilarity between
replication and transcriptionreplication and transcription
11. 11
replicationreplication transcriptiontranscription
templatetemplate double strandsdouble strands single strandsingle strand
substratesubstrate dNTPdNTP NTPNTP
primerprimer yesyes nono
EnzymeEnzyme DNA polymeraseDNA polymerase RNA polymeraseRNA polymerase
productproduct dsDNAdsDNA ssRNAssRNA
base pairbase pair A-A-TT, G-C, G-C A-A-UU, T-A, G-C, T-A, G-C
Differences betweenDifferences between
replication and transcriptionreplication and transcription
13. 13
The whole genome of DNA needs toThe whole genome of DNA needs to
be replicated, but only small portionbe replicated, but only small portion
of genome is transcribed in responseof genome is transcribed in response
to the development requirement,to the development requirement,
physiological need andphysiological need and
environmental changes.environmental changes.
DNA regions that can be transcribedDNA regions that can be transcribed
into RNA are called structural genes.into RNA are called structural genes.
14. 14
§1.1 Template
The template strand is the strand
from which the RNA is actually
transcribed. It is also termed as
antisense strand.
The coding strand is the strand
whose base sequence specifies the
amino acid sequence of the encoded
protein. Therefore, it is also called as
sense strand.
15. 15
G C A G T A C A T G T C5' 3'
3' C G T C A T G T A C A G 5' template
strand
coding
strand
transcription
RNAG C A G U A C A U G U C5' 3'
16. 16
• Only the template strand is used for the
transcription, but the coding strand is
not.
• Both strands can be used as the
templates.
• The transcription direction on different
strands is opposite.
• This feature is referred to as the
asymmetric transcription.
Asymmetric transcription
19. 19
§1.2 RNA Polymerase
The enzyme responsible for the RNAThe enzyme responsible for the RNA
synthesis is DNA-dependent RNAsynthesis is DNA-dependent RNA
polymerase.polymerase.
The prokaryotic RNA polymerase is aThe prokaryotic RNA polymerase is a
multiple-subunit protein of ~480kD.multiple-subunit protein of ~480kD.
Eukaryotic systems have three kinds ofEukaryotic systems have three kinds of
RNA polymerases, each of which is aRNA polymerases, each of which is a
multiple-subunit protein and responsiblemultiple-subunit protein and responsible
for transcription of different RNAs.for transcription of different RNAs.
22. 22
core enzymeholoenzyme
Holoenzyme
The holoenzyme of RNA-pol inThe holoenzyme of RNA-pol in E.coliE.coli
consists of 5 different subunits:consists of 5 different subunits: αα22 ββ β′β′
ωωσσ..
ω
β′
β
αα
σ
23. 23
subunitsubunit MWMW functionfunction
αα 3651236512
Determine the DNA to beDetermine the DNA to be
transcribedtranscribed
ββ 150618150618 Catalyze polymerizationCatalyze polymerization
β′β′ 155613155613 Bind & open DNA templateBind & open DNA template
σσ 7026370263
Recognize the promoterRecognize the promoter
for synthesis initiationfor synthesis initiation
RNA-pol of E. Coli
24. 24
Rifampicin, a therapeutic drug forRifampicin, a therapeutic drug for
tuberculosis treatment, can bindtuberculosis treatment, can bind
specifically to thespecifically to the ββ subunit of RNA-subunit of RNA-
pol, and inhibit the RNA synthesis.pol, and inhibit the RNA synthesis.
RNA-pol of other prokaryoticRNA-pol of other prokaryotic
systems is similar to that ofsystems is similar to that of E. coliE. coli inin
structure and functions.structure and functions.
25. 25
RNA-polRNA-pol II IIII IIIIII
productsproducts 45S rRNA45S rRNA hnRNAhnRNA
5S rRNA5S rRNA
tRNAtRNA
snRNAsnRNA
SensitivitySensitivity
to Amanitinto Amanitin
NoNo highhigh moderatemoderate
RNA-pol of eukaryotes
Amanitin is a specific inhibitor of RNA-pol.
26. 26
Each transcriptable region is calledEach transcriptable region is called
operon.operon.
One operon includes several structuralOne operon includes several structural
genes and upstream regulatorygenes and upstream regulatory
sequences (or regulatory regions).sequences (or regulatory regions).
The promoter is the DNA sequence thatThe promoter is the DNA sequence that
RNA-pol can bind. It is the key point forRNA-pol can bind. It is the key point for
the transcription control.the transcription control.
§1.3 Recognition of Origins
28. 28
5'
3'
3'
5'
-50 -40 -30 -20 -10 1 10
start-10
region
T A T A A T
A T A T T A
(Pribnow box)
-35
region
T T G A C A
A A C T G T
Prokaryotic promoter
Consensus sequence
30. 30
The -35 region of TTGACA sequenceThe -35 region of TTGACA sequence
is the recognition site and theis the recognition site and the
binding site of RNA-pol.binding site of RNA-pol.
The -10 region of TATAAT is theThe -10 region of TATAAT is the
region at which a stable complex ofregion at which a stable complex of
DNA and RNA-pol is formed.DNA and RNA-pol is formed.
32. 32
General concepts
Three phases: initiation, elongation,Three phases: initiation, elongation,
and termination.and termination.
The prokaryotic RNA-pol can bind toThe prokaryotic RNA-pol can bind to
the DNA template directly in thethe DNA template directly in the
transcription process.transcription process.
The eukaryotic RNA-pol requires co-The eukaryotic RNA-pol requires co-
factors to bind to the DNA templatefactors to bind to the DNA template
together in the transcription process.together in the transcription process.
33. 33
§2.1 Transcription of Prokaryotes
Initiation phase: RNA-polInitiation phase: RNA-pol recognizes therecognizes the
promoter and starts the transcription.promoter and starts the transcription.
Elongation phase: the RNA strand isElongation phase: the RNA strand is
continuously growing.continuously growing.
Termination phase: the RNA-pol stopsTermination phase: the RNA-pol stops
synthesis and the nascent RNA issynthesis and the nascent RNA is
separated from the DNA template.separated from the DNA template.
34. 34
a. Initiation
RNA-pol recognizesRNA-pol recognizes the TTGACAthe TTGACA
region, and slides to the TATAATregion, and slides to the TATAAT
region, thenregion, then opens the DNA duplex.opens the DNA duplex.
The unwound region is about 17The unwound region is about 17±±11
bp.bp.
35. 35
The first nucleotide on RNA transcriptThe first nucleotide on RNA transcript
is always purine triphosphate. GTP isis always purine triphosphate. GTP is
more often than ATP.more often than ATP.
The pppGpN-OH structure remains onThe pppGpN-OH structure remains on
the RNA transcript until the RNAthe RNA transcript until the RNA
synthesis is completed.synthesis is completed.
The three molecules form aThe three molecules form a
transcription initiation complex.transcription initiation complex.
RNA-pol (α2ββ′σ) - DNA - pppGpN- OH 3′
36. 36
No primer is needed for RNANo primer is needed for RNA
synthesis.synthesis.
TheThe σσ subunit falls off from the RNA-subunit falls off from the RNA-
pol once the first 3pol once the first 3′′,5,5′′ phosphodiesterphosphodiester
bond is formed.bond is formed.
The core enzyme moves along theThe core enzyme moves along the
DNA template to enter the elongationDNA template to enter the elongation
phase.phase.
37. 37
b. Elongation
• The release of the σ subunit causes
the conformational change of the
core enzyme. The core enzyme
slides on the DNA template toward
the 3′ end.
• Free NTPs are added sequentially to
the 3′ -OH of the nascent RNA strand.
38. 38
• RNA-pol, DNA segment of ~40nt and
the nascent RNA form a complex
called the transcription bubble.
• The 3′ segment of the nascent RNA
hybridizes with the DNA template,
and its 5′ end extends out the
transcription bubble as the synthesis
is processing.
45. 45
c. Termination
The RNA-pol stops moving on theThe RNA-pol stops moving on the
DNA template.DNA template. The RNA transcriptThe RNA transcript
falls off from the transcriptionfalls off from the transcription
complex.complex.
The termination occurs in eitherThe termination occurs in either ρρ
-dependent or-dependent or ρρ -independent-independent
manner.manner.
46. 46
The termination function of ρ factor
TheThe ρρ factor,factor, a hexamer, is aa hexamer, is a ATPaseATPase
and aand a helicasehelicase..
47. 47
ρρ-independent termination-independent termination
• The termination signal is a stretch of
30-40 nucleotides on the RNA
transcript, consisting of many GC
followed by a series of U.
• The sequence specificity of this
nascent RNA transcript will form
particular stem-loop structures to
terminate the transcription.
50. 50
The stem-loop structure alters theThe stem-loop structure alters the
conformation of RNA-pol, leading toconformation of RNA-pol, leading to
the pause of the RNA-pol moving.the pause of the RNA-pol moving.
Then the competition of the RNA-Then the competition of the RNA-
RNA hybrid and the DNA-DNA hybridRNA hybrid and the DNA-DNA hybrid
reduces the DNA-RNA hybridreduces the DNA-RNA hybrid
stability, and causes thestability, and causes the
transcription complex dissociated.transcription complex dissociated.
Among all the base pairings, theAmong all the base pairings, the
most unstable one is rU:dA.most unstable one is rU:dA.
Stem-loop disruptionStem-loop disruption
51. 51
§2.2 Transcription of Eukaryotes
Transcription initiation needsTranscription initiation needs
promoter and upstream regulatorypromoter and upstream regulatory
regions.regions.
The cis-acting elementsThe cis-acting elements are theare the
specific sequences on the DNAspecific sequences on the DNA
template that regulate thetemplate that regulate the
transcription of one or more genes.transcription of one or more genes.
a. Initiation
54. 54
RNA-pol does not bind the promoterRNA-pol does not bind the promoter
directly.directly.
RNA-pol II associates with sixRNA-pol II associates with six
transcription factors, TFII A - TFII H.transcription factors, TFII A - TFII H.
The trans-acting factors areThe trans-acting factors are thethe
proteins that recognize and bindproteins that recognize and bind
directly or indirectly cis-actingdirectly or indirectly cis-acting
elements and regulate its activity.elements and regulate its activity.
Transcription factors
56. 56
TBP of TFII D binds TATATBP of TFII D binds TATA
TFII A and TFII B bind TFII DTFII A and TFII B bind TFII D
TFII F-RNA-pol complex binds TFII BTFII F-RNA-pol complex binds TFII B
TFII F and TFII E open the dsDNATFII F and TFII E open the dsDNA
(helicase and ATPase)(helicase and ATPase)
TFII H: completion of PICTFII H: completion of PIC
Pre-initiation complex (PIC)
58. 58
TF II H is of protein kinase activity toTF II H is of protein kinase activity to
phosphorylate CTD of RNA-pol.phosphorylate CTD of RNA-pol. (CTD(CTD
is the C-terminal domain of RNA-pol)is the C-terminal domain of RNA-pol)
Only theOnly the pp--RNA-pol can move towardRNA-pol can move toward
the downstream, starting thethe downstream, starting the
elongation phase.elongation phase.
Most of the TFs fall off from PICMost of the TFs fall off from PIC
during the elongation phase.during the elongation phase.
Phosphorylation of RNA-pol
59. 59
The elongation is similar to that ofThe elongation is similar to that of
prokaryotes.prokaryotes.
The transcription and translation doThe transcription and translation do
not take place simultaneously sincenot take place simultaneously since
they are separated by nuclearthey are separated by nuclear
membrane.membrane.
b. Elongation
61. 61
• The termination sequence is
AATAAA followed by GT repeats.
• The termination is closely related to
the post-transcriptional modification.
c. Termination
64. 64
The nascent RNA, also known asThe nascent RNA, also known as
primary transcript, needs to beprimary transcript, needs to be
modified to become functionalmodified to become functional
tRNAs, rRNAs, and mRNAs.tRNAs, rRNAs, and mRNAs.
The modification is critical toThe modification is critical to
eukaryotic systems.eukaryotic systems.
66. 66
Primary transcripts of mRNA are called asPrimary transcripts of mRNA are called as
heteronuclear RNA (hnRNA).heteronuclear RNA (hnRNA).
hnRNA are larger than matured mRNA byhnRNA are larger than matured mRNA by
many folds.many folds.
Modification includesModification includes
Capping at the 5Capping at the 5′′- end- end
Tailing at the 3Tailing at the 3′′- end- end
mRNA splicingmRNA splicing
RNA editionRNA edition
§3.1 Modification of hnRNA
69. 69
The 5The 5′′- cap structure is found on- cap structure is found on
hnRNA too.hnRNA too. ⇒⇒ The capping processThe capping process
occurs in nuclei.occurs in nuclei.
The cap structure of mRNA will beThe cap structure of mRNA will be
recognized by the cap-binding proteinrecognized by the cap-binding protein
required for translation.required for translation.
The capping occurs prior to theThe capping occurs prior to the
splicing.splicing.
70. 70
b. Poly-A tailing at 3b. Poly-A tailing at 3′′ - end- end
There is no poly(dT) sequence on theThere is no poly(dT) sequence on the
DNA template.DNA template. ⇒⇒ The tailing processThe tailing process
dose not depend on the template.dose not depend on the template.
The tailing process occurs prior toThe tailing process occurs prior to
the splicing.the splicing.
The tailing process takes place in theThe tailing process takes place in the
nuclei.nuclei.
71. 71
The matured mRNAs are much shorter than
the DNA templates.
DNA
mRNA
c. mRNA splicingc. mRNA splicing
72. 72
A~G no-coding region 1~7 coding region
L 1 2 3 4 5 6 7
7 700 bp
The structural genes are composed ofThe structural genes are composed of
coding and non-coding regions thatcoding and non-coding regions that
are alternatively separated.are alternatively separated.
Split geneSplit gene
EEAA BB CC DD FF GG
73. 73
Exon and intronExon and intron
Exons are the coding sequences that
appear on split genes and primary
transcripts, and will be expressed to
matured mRNA.
Introns are the non-coding sequences
that are transcripted into primary
mRNAs, and will be cleaved out in the
later splicing process.
77. 77
U pA G pU5' 3'
5'exon 3'exon
intron
pG-OH
pGpA
G pU 3'U5' OH
first transesterification
Twice transesterificationTwice transesterification
second transesterification
U5' pU 3'
pGpA
GOH
5'
3'
78. 78
Taking place at the transcriptionTaking place at the transcription
levellevel
One gene responsible for more thanOne gene responsible for more than
one proteinsone proteins
Significance: gene sequences, afterSignificance: gene sequences, after
post-transcriptional modification,post-transcriptional modification,
can be multiple purposecan be multiple purpose
differentiation.differentiation.
d. mRNA editing
79. 79
Different pathway of apo B
Human apo B
gene
hnRNA (14 500 base)
liver
apo B100
（ 500 kD ） intestine
apo B48
（ 240 kD ）
CAA to UAA
At 6666
85. 85
§3.3 Modification of rRNA
45S transcript in nucleus is the45S transcript in nucleus is the
precursor of 3 kinds of rRNAs.precursor of 3 kinds of rRNAs.
The matured rRNA will be assembledThe matured rRNA will be assembled
with ribosomal proteins to formwith ribosomal proteins to form
ribosomes that are exported toribosomes that are exported to
cytosolic space.cytosolic space.
87. 87
The rRNA precursor of tetrahymenaThe rRNA precursor of tetrahymena
has the activity of self-splicinghas the activity of self-splicing
(1982).(1982).
The catalytic RNA is called ribozyme.The catalytic RNA is called ribozyme.
Self-splicing happened often forSelf-splicing happened often for
intron I and intron II.intron I and intron II.
§3.4 Ribozyme
88. 88
• Both the catalytic domain and the
substrate locate on the same
molecule, and form a hammer-head
structure.
• At least 13 nucleotides are conserved.
90. 90
Be a supplement to the centralBe a supplement to the central
dogmadogma
Redefine the enzymologyRedefine the enzymology
Provide a new insights for the originProvide a new insights for the origin
of lifeof life
Be useful in designing the artificialBe useful in designing the artificial
ribozymes as the therapeuticalribozymes as the therapeutical
agentsagents
Significance of ribozyme
