Transcription is the process of synthesizing RNA using a DNA template. There are four main types of RNA - mRNA, tRNA, rRNA and snRNA. Transcription involves initiation, elongation and termination. In initiation, RNA polymerase binds to a promoter and transcription begins. In elongation, RNA is continuously synthesized using the DNA as a template. Termination occurs when RNA polymerase stops moving along the DNA template. Eukaryotic transcription requires transcription factors to help RNA polymerase bind DNA, while prokaryotic transcription involves direct binding of RNA polymerase to DNA. The nascent RNA transcript undergoes processing including capping, polyadenylation, splicing and editing to become a mature RNA.

1. Transcription
(RNA
biosynthesis)

2. Transcription
The synthesis of RNA m olecules using DNA
strands as the templates so that the genetic
information can be transferred from DNA to
RNA.

3. Overview
There are four major types of RNA molecules:
a. Messenger RNA (mRNA) encodes the amino
acid sequence of a polypeptide.
b. Transfer RNA (tRNA) brings amino acids to
ribosomes during translation.
c. Ribosomal RNA (rRNA) combines with
proteins to form a ribosome, the catalyst for
translation.
d. Small nuclear RNA (snRNA) combines with
proteins to form complexes used in eukaryotic
RNA processing.

4. The Transcription Process RNA
Synthesis
1. Transcription, or gene expression, is regulated by gene
regulatory elements associated with each gene.
2. DNA unwinds in the region next to the gene.
3. RNA is transcribed 5’-to-3’. The template DNA strand is
read 3’-to-5’. Its complementary DNA, the nontemplate
strand, has the same polarity as the RNA.
4. RNA polymerization is similar to DNA synthesis, except:
a. The precursors are NTPs (not dNTPs).
b. No primer is needed to initiate synthesis.
d. Uracil is inserted instead of thymine.

5. Similarity between
replication and transcription
• Both processes use DNA as the template.
• Phosphodiester bonds are formed in both
cases.
• Both synthesis directions are from 5´ to 3´.

6. The RNA polymerase-catalyzed synthesis of
RNA on a DNA template strand

7. Differences between
replication and transcription
replication transcription
template double strands single strand
substrate dNTP NTP
primer yes no
Enzyme DNA polymerase RNA polymerase
product dsDNA ssRNA
base pair A-T, G-C A-U, T-A, G-C

8. The Transcription Process
Initiation of Transcription at Promoters
Transcription is divided into three steps for both
prokaryotes and eukaryotes.
initiation, elongation and termination.
The process of elongation is highly conserved
between prokaryotes and eukaryotes, but
initiation and termination are somewhat
different.

9. Template and Enzymes

10. • The whole genome of DNA needs to be
replicated, but only small portion of genome
is transcribed in response to the
development requirement, physiological need
and environmental changes.
• DNA regions that can be transcribed into
RNA are called structural genes.

11. 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.
coding
strand
G C 5' A G T A C A T G T C 3'
3' C G T C A T G T A C A G 5' template
strand
transcription
5' G C A G U A C A U G U C 3' RNA

12. Asymmetric transcription
• Only the template strand is used for the transcription, but
the coding strand is not.
• The transcription direction on different strands is
opposite.
• This feature is referred to as the asymmetric
transcription.
5'
3'
3'
5'

13. Organization of coding information in
the adenovirus genome

14. RNA Polymerase
• The enzyme responsible for the RNA
synthesis is DNA-dependent RNA
polymerase.
– The prokaryotic RNA polymerase is a
multiple-subunit protein of ~480kD.
– Eukaryotic systems have three kinds of
RNA polymerases, each of which is a
multiple-subunit protein and responsible for
transcription of different RNAs.

15. RNA-pol of E. Coli
The holoenzyme of RNA-p ol in E.coli consists of 5
different subunits: a2 b b¢ ws.
core enzyme
b¢ b
a a
w
s
subunit MW function
a 36512 Determine the DNA to be
transcribed
b 150618 Catalyze polymerization
b¢ 155613 Bind & open DNA template
s 70263 Recognize the promoter
for synthesis initiation

16. • Rifampicin, a therapeutic drug for
tuberculosis treatment, can bind
specifically to the b subunit of RNA-pol,
and inhibit the RNA synthesis.
• RNA-pol of other prokaryotic systems is
similar to that of E. coli in structure and
functions.

17. RNA-pol of eukaryotes
RNA-pol I II III
products 45S rRNA hnRNA
5S rRNA
tRNA
snRNA
Sensitivity
to Amanitin No high moderate
Amanitin is a specific inhibitor of RNA-pol.
from a mushroom, inhibits Pol II, and Pol
III at higher concentrations.

18. Recognition of Origins
• Each transcriptable region is called
operon.
• One operon includes several structural
genes and upstream regulatory sequences
(or regulatory regions).
• The promoter is the DNA sequence that
RNA-pol can bind. It is the key point for
the transcription control.

19. 5'
3'
3'
5'
Promoter
regulatory
sequences structural gene
pRroNmA-opotol r

20. 5'
3'
3'
5'
Prokaryotic promoter
-50 -40 -30 -20 -10 1 10
-10 start
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
Consensus sequence

21. Consensus Sequence

22. • The -35 region of TTGACA sequence is the
recognition site and the binding site of RNA-pol.
• The -10 region of TATAAT is the region at
which a stable complex of DNA and RNA-pol
is formed.

23. Transcription Process

24. General concepts
• Three phases: initiation, elongation,
and termination.
• The prokaryotic RNA-pol can bind to
the DNA template directly in the
transcription process.
• The eukaryotic RNA-pol requires co-factors
to bind to the DNA template
together in the transcription process.

25. Transcription of Prokaryotes
• Initiation phase: RNA-pol recognizes
the promoter and starts the
transcription.
• Elongation phase: the RNA strand is
continuously growing.
• Termination phase: the RNA-pol stops
synthesis and the nascent RNA is
separated from the DNA template.

26. a. Initiation
• RNA-pol recognizes the TTGACA
region, and slides to the TATAAT
region, then opens the DNA duplex.
• The unwound region is about 17±1
bp.

27. • The first nucleotide on RNA transcript
is always purine triphosphate. GTP is
more often than ATP.
• The pppGpN-OH structure remains on
the RNA transcript until the RNA
synthesis is completed.
• The three molecules form a
transcription initiation complex.
RNA-pol (a2bb¢s) - DNA - pppGpN- OH 3¢

28. • No primer is needed for RNA
synthesis.
• The s subunit falls off from the RNA-pol
once the first 3¢,5¢ phosphodiester
bond is formed.
• The core enzyme moves along the
DNA template to enter the elongation
phase.

29. b. Elongation
• The release of the s 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.

30. • RNA-pol, DNA seg ment 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.

31. Transcription bubble

32. RNA-pol of E. Coli

33. RNA-pol of E. Coli

36. Simultaneous
transcriptions and
translation

37. c. Termination
• The RNA-pol stops moving on the
DNA template. The RNA transcript
falls off from the transcription
complex.
• The termination occurs in either r
-dependent or r -independent
manner.

39. The termination function of r factor
The r factor, a hexamer, is a ATPase
and a helicase.

40. r-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.

41. DNA
5¢TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3¢
5¢TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3¢
RNA
UUUU...…
UUUU...…

44. Stem-loop disruption
• The stem-loop structure alters the
conformation of RNA-pol, leading to
the pause of the RNA-pol moving.
• Then the competition of the RNA-RNA
hybrid and the DNA-DNA hybrid
reduces the DNA-RNA hybrid
stability, and causes the
transcription complex dissociated.
• Among all the base pairings, the
most unstable one is rU:dA.

45. Transcription of Eukaryotes
a. Initiation
• Transcription initiation needs
promoter and upstream regulatory
regions.
• The cis-acting elements are the
specific sequences on the DNA
template that regulate the
transcription of one or more genes.

46. Cis-acting element
structural gene
cis-acting element
GCGC CAAT TATA
exon intron exon
start
CAAT box
enhancer
GCbox
TATA box (Hogness box)

47. TATA box

48. Transcription factors
• RNA-pol does not bind the promoter
directly.
• RNA-pol II associates with six
transcription factors, TFII A - TFII H.
• The trans-acting factors are the
proteins that recognize and bind
directly or indirectly cis-acting
elements and regulate its activity.

49. TF for eukaryotic transcription

50. Pre-initiation complex (PIC)
• TBP of TFII D binds TATA
• TFII A and TFII B bind TFII D
• TFII F-RNA-pol complex binds TFII B
• TFII F and TFII E open the dsDNA (helicase and ATPase)
• TFII H: completion of PIC
RNA pol II
TF II F
TBP TAF
TATA
DNA
TF II
A
TF II
B
TF II E
TF II H

51. Transcription factors and the pre-initiation complex

52. Phosphorylation of RNA-pol
• TF II H is of protein kinase activity to
phosphorylate CTD of RNA-pol. (CTD
is the C-terminal domain of RNA-pol)
• Only the RNA-pol can move toward
the downstream, starting the
elongation phase.
• Most of the TFs fall off from PIC
during the elongation phase.

53. b. Elongation
• The elongation is similar to that of
prokaryotes.
• The transcription and translation do
not take place simultaneously since
they are separated by nuclear
membrane.

55. RNA-Pol
nucleosome
RNA-Pol
RNA-Pol
moving
direction

56. c. Termination
• When the RNA Polymerase transcribes the
terminator region of the DNA, the polymerase
releases the mRNA
• The termination sequence is AATAAA
followed by GT repeats.
• In eukaryotes, the release factor (eRF) which
recognizes all three stop codons. The overall
process of termination is similar in
prokaryotes, but 3 release factors exist, RF1,
RF and RF3.

58. Post-Transcriptional
Modification

59. • The nascent RNA, also known as
primary transcript, needs to be
modified to become functional
tRNAs, rRNAs, and mRNAs.
• The modification is critical to
eukaryotic systems.

60. Modification of hnRNA
• Primary transcripts of mRNA are called as
heteronuclear RNA (hnRNA).
• hnRNA are larger than matured mRNA by many
folds.
• Modification includes
– Capping at the 5¢- end
– Tailing at the 3¢- end
– mRNA splicing
– RNA editing

61. a. Capping at the 5¢- end
OH OH
O
H2N N
O P
CH3
O
CH2
O
N NH
N
O OH
O P
O
O
N
NH2
AAAAA-OH
O
Pi
5'
3'
O
N H2C
HN N
O
O
O
O P
O
O
O P
O
5'
m7GpppGp----

62. • The 5¢- cap structure is found on hnRNA
too. Þ The capping process occurs in
nuclei.
• The cap structure of mRNA will be
recognized by the cap-binding protein
required for translation.
• The capping occurs prior to the splicing.
addition of 5’cap:
Prevents “unraveling”
Helps ribosome attach

64. b. Poly-A tailing at 3¢ - end
• There is no poly(dT) sequence
on the DNA template. Þ The
tailing process dose not depend
on the template.
• The tailing process occurs prior
to the splicing.
• The tailing process takes place
in the nuclei.
addition of poly A tail
Prevents “unraveling”
Assists in the export of mRNA from
nucleus

65. c. mRNA splicing
DNA
mRNA
The matured mRNAs are much shorter than
the DNA templates.

66. Split gene
The structural genes are composed of
coding and non-coding regions that
are alternatively separated.
7 700 bp
L 1 2 3 4 5 6 7
A B C D E F G
A~G no-coding region 1~7 coding region

67. Exon 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.

68. mRNA splicing

69. Splicing mechanism

70. lariat

71. TGGCNNAGTGC GGTTCGANNCC
RNA-pol III
tRNA precursor
DNA
Precursor transcription

72. Twice transesterification
intron
5'exon 3'exon
5' U pA G pU 3'
pG-OH
pGpA
first transesterification
5' UOH G pU 3'
second transesterification
pGpA
5' U pU 3'
GOH
5'
3'

73. d. mRNA editing
• Taking place at the transcription
level
• One gene responsible for more than
one proteins
• Significance: gene sequences, after
post-transcriptional modification,
can be multiple purpose
differentiation.

74. Different pathway of apo B
Human apo B
gene
hnRNA (14 500 base)
CAA to UAA
liver
apo B100
（500 kD） intestine
apo B48
（240 kD）

75. Modification of tRNA

76. Cleavage
RNAase P
endonuclease
ligase

77. Addition of CCA-OH
tRNA nucleotidyl
transferase
ATP ADP

78. Base modification
（1）
（1）
（3）
（2）
（4）
1. Methylation
A→mA, G→mG
2. Reduction
U→DHU
3. Transversion
U→ψ
4. Deamination
A→I
Little is known about the role of specific base modifications of transfer RNAs

79. Modification of rRNA
• 45S transcript in nucleus is the precursor of 3 kinds
of rRNAs.
• The matured rRNA will be assembled with ribosomal
proteins to form ribosomes that are exported to
cytosolic space.
rRNA
18S 5.8S 28S
transcription
splicing
45S-rRNA
18S-rRNA
5.8S and 28S-rRNA

80. Prokaryotic Regulation
• Control of transcription initiation can be:
– positive control – increases transcription when
activators bind DNA
– negative control – reduces transcription when
repressors bind to DNA regulatory regions
called operators

81. Prokaryotic Regulation
• Prokaryotic cells often respond to their environment by
changes in gene expression.
• Genes involved in the same metabolic pathway are
organized in operons.
• A regulatory sequence adjacent to such a unit determines
whether it is transcribed - this is the ‘operator’
• Some operons are induced when the metabolic pathway is
needed.
• Some operons are repressed when the metabolic pathway
is no longer needed.

82. Prokaryotic Regulation
• The lac operon contains genes for the use of
lactose as an energy source.
• Regulatory regions of the operon include the CAP
(catabolite activator protein) binding site,
promoter, and the operator.
• The coding region contains genes for 3 enzymes:
b-galactosidase, permease, and transacetylase

84. Prokaryotic Regulation
• The lac operon is negatively regulated by a
repressor protein:
– lac repressor binds to the operator to block
transcription
– in the presence of lactose, an inducer molecule
binds to the repressor protein
– repressor can no longer bind to operator
– transcription proceeds

87. Prokaryotic Regulation
• In the presence of both glucose and lactose,
bacterial cells prefer to use glucose.
• Glucose prevents induction of the lac operon.
– binding of CAP – cAMP complex to the CAP
binding site is required for induction of the lac operon
– high glucose levels cause low cAMP levels
– high glucose  low cAMP  no induction

90. Prokaryotic Regulation
• The trp operon encodes genes for the
biosynthesis of tryptophan.
• The operon is not expressed when the cell
contains sufficient amounts of tryptophan.
• The operon is expressed when levels of
tryptophan are low.

91. Prokaryotic Regulation
• The trp operon is negatively regulated by
the trp repressor protein
– trp repressor binds to the operator to block
transcription
– binding of repressor to the operator requires a
corepressor which is tryptophan
– low levels of tryptophan prevent the repressor
from binding to the operator

94. Eukaryotic Regulation
• Controlling the expression of eukaryotic
genes requires transcription factors.
– general transcription factors are required for
transcription initiation
• required for proper binding of RNA polymerase to
the DNA
– specific transcription factors increase
transcription in certain cells or in response to
signals

95. Eukaryotic Transcription
• General transcription factors bind to the promoter region of
the gene.
• RNA polymerase II then binds to the promoter to begin
transcription at the start site (+1).
• Enhancers are DNA sequences to which specific
transcription factors (activators) bind to increase the rate of
transcription.

96. Mechanisms of enhancer action
• DNA looping model postulates that proteins bound to a distant
enhancer interact directly with components of the transcription
initiation complex, by looping out the DNA
• An enhancer noncovalently linked to a promoter via a protein bridge
is functional
• Enhancer function requires close proximity to the promoter
• Enhancers do not serve as entry sites for RNA polymerase II

98. Eukaryotic Transcription
• Coactivators and mediators are also
required for the function of transcription
factors.
– coactivators and mediators bind to transcription
factors and bind to other parts of the
transcription apparatus

100. Posttranscriptional Regulation
• Control of gene expression usually involves the
control of transcription initiation.
• But gene expression can be controlled after
transcription, with mechanisms such as:
– RNA interference
– alternative splicing
– RNA editing
– mRNA degradation

101. Posttranscriptional Regulation
• RNA interference involves the use of small
RNA molecules
• The enzyme Dicer chops double stranded
RNA into small pieces of RNA
– micro-RNAs bind to complementary RNA to
prevent translation
– small interfering RNAs degrade particular
mRNAs before translation

102. Micro RNA (miRNA)
• Production of a functional miRNA begins in the
nucleus
• Ends in the cytoplasm with a ~22 nt RNA that
functions to repress gene expression
• miRNA loaded into RNA induced silencing
complex (RISC)
• RISC is targeted to repress the expression of genes
based on sequence complementarity to the miRNA

103. Cytoplasm
Dicer
Pri-microRNA
Mature miRNA
RISC mRNA
RNA Polymerase II
microRNA gene
RISC
mRNA cleavage
mRNA
RISC RISC
Inhibition of translation
Nucleus
Drosha
Exportin 5
Pre-microRNA

104. siRNA
• RNA interference involves the production
of siRNAs
• Production similar to miRNAs but siRNAs
arise from long double-stranded RNA
• Dicer cuts yield multiple siRNAs to load
into RISC (RNA-induced silencing
complex)
• Target mRNA is cleaved

105. miRNA or siRNA?
• Biogenesis of both miRNA and siRNA
involves cleavage by Dicer and
incorporation into a RISC complex
• Main difference is target
– miRNA repress genes different from their origin
– Endogenous siRNAs tend to repress genes that they
were derived from

106. Exogenous dsRNA, transposon, virus
P
P
Repeated cutting
by dicer
P
P
P
P
P
P siRNAs
Ago +
RISC
Ago
RISC
siRNA
in RISC
Cleavage of target mRNA
mRNA

107. Alternative splicing
• Introns are spliced out of pre-mRNAs to produce
the mature mRNA
• Tissue-specific alternative splicing
• The mature mRNAs in each tissue possess
different exons, resulting in different
polypeptide products from the same gene.
• Determined by tissue-specific factors that
regulate the processing of the primary transcript

109. Why bother with introns?
• Introns may regulate gene activity and the
passage of mRNA into the cytoplasm
• Genes may play roles in multiple proteins,
introns may enable a gene to be diverse in
function
• May increase recombination of genetic
material (easier to cut and paste)

110. RNA editing
• Creates mature mRNA that are not truly
encoded by the genome
• Involves chemical modification of a base to
change its base-pairing properties
• Apolipoprotein B exists in 2 isoforms
– One isoform is produced by editing the mRNA
to create a stop codon
– This RNA editing is tissue-specific

111. • Initiation of translation can be controlled
– Ferritin mRNA only translated if iron present
• Mature mRNA molecules have various
half-lives depending on the gene and the
location (tissue) of expression
– Target near poly-A tail can cause loss of the tail and
destabilization
•The amount of polypeptide produced from a particular
gene can be influenced by the half-life of the mRNA
molecules.

112. DNA
2. RNA splicing
Gene expression
can be controlled
by altering the
rate of splicing in
eukaryotes.
Alternative splicing
can produce
multiple mRNAs
from one gene.
5´ cap
Cut
intron
3´ poly-A tail
Mature RNA transcipt Exons
Introns
RNA polymerase II
3´
5´ Primary RNA transcript
1. Initiation of
transcription
Most control of
gene expression
is achieved
by regulating
the frequency
of transcription
initiation.

113. 3. Passage through
the nuclear
membrane
Gene expression
can be regulated
by controlling
access to or
efficiency of
transport channels.
4. Protein synthesis
Many proteins take
part in the
translation process,
and regulation
of the availability
of any of them alters
the rate of gene
expression by
speeding or slowing
protein synthesis.
3´
5´
Nuclear
pore
Small
subunit
5´ cap
mRNA
Large
3´ poly-A tail subunit

114. 5. RNA interference
Gene expression
is regulated by
small RNAs. Protein
complexes
containing siRNA
and miRNA target
specific mRNAs for
destruction or inhibit
their translation.
Completed
polypeptide
chain
6. Posttranslational
modification
Phosphorylation
or other chemical
modifications can
alter the activity
of a protein after
it is produced.
RISC
P
P

115. Ribozyme
• The rRNA precursor of tetrahymena
has the activity of self-splicing.
• The catalytic RNA is called ribozyme.
• Self-splicing happened often for intron I
and intron II.
• Both the catalytic domain and the
substrate locate on the same molecule,
and form a hammer-head structure.
• At least 13 nucleotides are conserved.

116. Hammer-head

117. Significance of ribozyme
• Be a supplement to the central dogma
• Redefine the enzymology
• Provide a new insights for the origin of
life
• Be useful in designing the artificial
ribozymes as the therapeutical agents

118. Retrovirus Replication Cycle
ds DNA
host DNA Provirus
Packaging into virus ; budding

119. Sample questions
• Which of the following enzyme is used for
synthesis of RNA under the direction of
DNA?
• A. RNA polymerase
• B. DNA ligase
• C. DNA polymerase
• D. RNA ligase

120. • Which of the following is a product of
transcription?
• A. mRNA
• B. tRNA
• C. rRNA
• D. all of these

121. • Recognition/binding site of RNA
polymerase is called
• A. receptor
• B. promoter
• C. facilitator
• D. terminator

122. • An mRNA transcript of a gene contains
• A. a start codon
• B. a stop codon
• C. a terminator
• D. all of these

123. • The components found in all prokaryotic
transcription terminators is
• A. a poly-U region
• B. Rho factor
• C. a hairpin structure
• D. none of these

124. • Where in the cell is the DNA transcribed into
mRNA?
• A.Cytoplasm
• B. Nucleus
• C. Golgi
• D.Cell cytoskeleton

125. • Which of the following does NOT happen during hnRNA
processing?
• A. Introns are spliced out.
• B. A 7-methylguanosine cap is added to the 5' end of the RNA.
• C. A poly A tail is added.
• D. Ribosomes bind and begin translation.

126. • Since the two strands of the DNA molecule are
complementary, for any given gene:
• A. The RNA polymerase can bind to either strand.
• B. Only one strand actually carries the genetic code for a
particular gene.
• C. Each gene possesses an exact replica so that no
mutation occurs.
• D. A gene transcribed in the 5’ to 3’ direction on one
strand can be transcribed in the 3’ to 5’ direction on the
other strand.