? G1 RNA structure
? G2 Transcription in prikaryotes
? G3 The lac operon
? G4 The trp operon
? G5 Transcription in eukaryotes
? G6 Transcription of protein-coding genes
? G7 Regulation of transcription by RNA pol II
? G8 Processing of eukaryotic pre-mRNA
? G9 Ribosomal RNA
? G10 Transfer RNA
G RNA synthesis and processing
Central Dogma
RNA
DNA
Protein
Transcription
Translation
Replication
Reverse
Transcription
Juang RH (2004) BCbasics
Nucleotide
Nucleoside
Phosphoric acid
Pentose
Bases
Bases
Purine
Pyrimidine
A
G
C
U
G1 RNA structure
Nucleic acid
Infrequent bases
? s4U 4-硫尿嘧啶
? Inosine 次黄嘌呤
? m1G 1-甲基鸟嘌呤
? m1A 1-甲基腺嘌呤
? DHU 5,6-二氢尿嘧啶
? s2C 2-硫胞嘧啶
? Pseudouridine(ψ )假尿嘧啶,C1-C5
Rare bases
Nucleoside(C1-N1 or C1-N9)
? Adenosine
? Guanosine
? Cytidine
? Uridine
? Pseudouridine(ψ):C1-C5
Covalent structure
? long polymer chain
? ATP,UTP,GTP,CTP
? 3’-5’ phosphodiester bonds（ not 2’-5’）
? 5’-GUCATUATCCA-3’
Long polymer chain
断裂
RNase
微碱
Difference between RNA and DNA
RNA secondary structure
? RNA molecules are largely single-stranded but there
are regions of self-complementary where the RNA
chain forms internal double-stranded regions
RNA secondary structure
RNA
RNA
tRNA secondary structrue
tRNA
tRNA
? Three phases of transcription
1,Promoters and initiation起始
2,Elongation延伸
3,Termination终止
? RNA processing加工
G2 transcription in prokaryotes
3’ 5’
5’ 3’ RNA
template strand
nontemplate strand (+) strand
(-) strand
5’ 3’
coding strand
AUG
ATG
TAC
UAG
TAG
ATC
Start
codon
Same
sequence
Template strand
Sense(+) strand=coding strand
Antisense(-) strand=template strand
Transcript
5’ 3’
RNA
3’ 5’
DNA
3’ 5’
RNA
5’ 3’
DNA
Promotor
RNA synthesis
RNA polymerase
RNA polymerase
Promoter
? During initiation,RNA polymerases recognizes a
specific site on the DNA,upstream from the gene
that will be transcribed,called a promoter site,
? 启动子是指 RNA 聚合酶识别、结合和开始转录的一
段 DNA序列。
-10sequence:TATAAT(Pribnow box):recognition site
-35sequence:TTGACA is important in DNA unwind
16-18bp
5-8bp
Promoters differ by up to 1000-fold in their
efficiency of initiation of transcription
Promoter
Promoters and initiation
Transcription bubble
Direction of transcription
Elongation
Terminator
Rho depentend terminator
RNA processing
? Induction of the lac operon
? The lac repressor
? CRP/CAP
? Positive and negative regulation
G3 the lac operon
The Lac operon
The lac operon
? The operon is transcribed by RNA polymerase to
produce a single polycistronic mRNA that is then
translated to produce all three enzymes
? These enzymes are involved in lactose metabolism,
? When lactose is absent,E.coli makes only small amounts
of these enzyme,but the presence of lactose induces
synthesis of large amounts of all three enzymes,
3 Structure genes
Lac Z:β-Galactosidase( β-半乳糖苷酶 ),which hydrolyzes
lactose(乳糖 )to glucose(葡萄糖 )and galactose(半乳糖 )
Lac Y:galactoside Permease (β-半乳糖苷透性酶 ),it transports
lactose into the cell across the cell membrane
Lac A:thiogalactosideTransacelylase：硫代 半乳糖苷转乙酰基
酶
Hydrolysis
Induction of the lac operon
? The mechanism of induction is that background level of β
–galactosidase converts some lactose to allolactose异乳
糖 which then acts as an inducer and turns on
transcription of the lac operon,
? IPTG（ isopropylthiogalactoside） can also act as an
inducer,
IPTG
The lac repressor
? Transcription of the operon controlled by the lac
repressor protein encoded by the lac I gene
? The lacI gene has its own promoter to which RNA
polymerase binds and initiates transcription,
The lac repressor
The lac repressor
The inducer
CRP/ACP
? Catabolite activator protein,CAP is an activator required
for high level transcription of the lac operon,
? The active molecule is a CRP dimer that binds 3‘-5'
cAMP to form a CRP-cAMP complex,
? CRP-cAMP binds to the lac promoter and increases the
binding of RNA polymerase,stimulating transcription
of the lac operon,
CRP/ACP
? When glucose is present,the intracellular level of cAMP
falls,CRP cann’t bind to the lac promoter and the lac
operon is only weakly transcribed,
? When glucose is absent,the level of intracellular cAMP
rises,the CRP-cAMP complex stimulates transcription of
the lac operon and allows lactose to be used as an
alternative carbon source,
CRP/ACP
CRP/ACP
Positive and negative regulation
? In negative regulation of prokaryotic gene
expression,bound repressor prevents transcription of the
structural genes,
? In positive regulation of gene expression,an activator
binds to DNA and increnses the rate of transcription,
? Through the lac repressor and CRP/CAP protein,the lac
operon is subject to both negative and positive control,
? Organization of the trp operon
? Repression
? Attenuation
? Attenuation vs,repression
G4 The trp operon
Organization of the trp operon
? The trp operon contains five structural genes encoding
enzymes for tryptophan biosynthesis,a trp promoter and
a trp operator sequence,
The trp operon
? The operon is transcribed only when tryptophan is
scarce,
Repression
? When tryptophan is present,tryptophan synthesis is not
needsd,
? In this situation,acting as a corepressor,
tryptophan binds to the repressor and activates it so that
the repressor now binds to the trp operator and stops
transcription of the trp operon,
The trp operon
Attenuation弱化作用
? The trp operon is also controlled by attenuation,
? A leader sequence in the polycistronic mRNA upstream
of the coding region of the trpE structural gene encodes
a 14 amino acid leader peptide including two tryptophan
residues
Leader sequence
Attenuation
? The RNA leader sequence can for several possible stem-loop
secondary structures,one of which can act as a transcription
terminator whilst a different stem-loop can act as an anti-
terminator,
Attenuation
? In the presence of tryptophan,ribosomes bind to the trp
polycistronic mRNA that is being transcribed,following
closely behind the RNA polymerase and begin to
translate the leader sequence,
? When tryptophan is present,translation of the leader
sequence occurs to completion,
Attenuation of the operon
? If tryptophan is scarce,the ribosome pauses when
attempting to translate the two trp codons in the leader
sequence,which leaves the leader sequence available to
form the anti-terminator stem-loop,
? Transcription of the trp operon is then allowed to continue,
Attenuation of the operon
Attenuation vs repression
? The trp operon is regulated by both repression(which
determines whether transcription will occur or not) and
attenuation (which fine tunes transcription),
? Other operons for amino acid biosynthetic pathways may also
be regulated by both repression and attenuation or only by
attenuation,
? Three RNA polymerases
? RNA synthesis
? RNA polymerases subunits
G5 Transcription in eukaryotes
Three RNA polymerases
? RNA pol I is located in the nucleolus核仁 and transcribes
the 28S,18S and 5.8S rRNA
? RNA pol II is lacated in the nucleoplasm核质 and
transcribes pre-mRNA and snRNAs
? RNA pol III is lacated in the nucleoplasm and transcribes the
genes for tRNA,5S rRNA,U6 snRNA
Three RNA polymerases
RNA synthesis
Precursors:4 dNTPs(N=A,U,G,C) Mg2+
? Each RNA polymerase transcribes only one strand,the
antisense(-)strand of a double – stranded DNA
template,directed by a promoter,
? Synthesis occurs 5' → 3'and not require a primer,
RNA polymerase subunits
? Each of the three RNA polymerases contain 12 or more
subunits,some of which are similar to those of E.coli
RNA polymerases,
? However,four to seven subunits in each enzyme are
unique to that enzyme,
? Gene organization
? Initiation of transcription
? Elongation
? termination
G6 transcription of protein coding
genes in eukaryotes
Gene organization
? Most protein-coding genes in eukaryotes consist of coding
sequences called exons interrupted by noncoding sequences
called introns,
? The number of introns and their size varies from gene to gene,
? The primary transcript undergoes processing reactions to
yield mature mRNA,
mRNA
DNA
5’ 3’
cap
poly A
tail
exon exon exon
intron intron
mature mRNA
Processing
Transcription
Splicing
promotor
3’ 5’ start codon stop codon
to cytoplasm
move intron
3 exons of myoglobin
St
rye
r (
19
95
) B
ioch
em
ist
ry
(4e
) p
.15
3
Mathews et al (2000) Biochemistry (3e) p.231
Initiation of transcription
? Most promoter sites for RNA polymerase II have a TATA
box located about 25 bp upstream of the transcriptional
start site,
TATA box
Transcription factors
? RNA polymerase binding to the promoter requires the
formation of a transcription initiation complex involving
several general transcription factors that assemble in a
strict order,
Elongation and termination
? Elongation continues until transcription comes to a halt
at varying distances downstream of the gene,releasing
the primary RNA transcript,pre- mRNA,
? This molecule then undergoes processing reactions to
yield mRNA,
G7 regulation of transcription
by RNA pol II
? Mechanism of regulation
? Upstream of regulation
? Enhancers
? Transcription factors have multiple domains
? DNA binding domains
? Dimerization domains
? Activation domains
? repressors
Mechanism of regulation
? Transcriptional regulation occurs via transcription factors
that bind to short control elements associated with the
target genes and then interact with each other and with
the transcription initiation complex to increase or
decrease the rate of transcription of the target gene,
NOTES
Transcription factors：与转录控制有关的蛋白质
Cis-acting elements(顺式作用元件 ),转录因子识别并结合的
DNA调控区。
Tans-acting factors(反式作用元件 )：与顺式元件结合的蛋白
质转录因子。
Enhancers(增强子 )：促进启动子的转录活性的一类顺式元件
Upstream regulatory elements
? Many transcription factors bind to control elements
located upstream within a few hundred base-pairs of the
protein-coding gene,
? The SP1 box and CAAT box are examples of such
regulatory elements found upstream of most protein-
coding genes,but some upstream regulatory elements
are associated with only a few genes and are
responsible for gene-specific transcriptional regulation,
The SP1 box and CAAT box
Enhancers
? positive transcriptional control elements
? 100-200 bp
? upstream or downstream,
? active in a long distance away,
? The transcription factors bound to these long-distance
elements interact with the transcription initiation complex
by looping out of the DNA,
Enhancers
Transcription factors have
multiple domains
? A DNA-binding domain that recognizes the specific DNA
control element to bind to
? An activation domain that interacts with other
transcription factors or the RNA polymerases,
Helix-turn-helix
Helix-turn-helix
Helix-turn-helix
Zinc finger
Zinc finger
Zinc finger
A leucine zippers
Leu zipper
A leucine zippers
Helix-loop-helix motif
Helix-loop-helix motif
Activation domains
? No common structural motifs are known for the
activation domains of transcription factors,
1,Acid Activation domains,rich in acidic amino acids
2,Glutamine-rich domains
3,Proline-rich domains
Repressors
? Repressor proteins that inhibit the transcription of
specific genes in eukaryotes may bind either to control
elements near to the target gene or to silencers that may
be located a long distance near to the target gene,
? The repressor may inhibit transcription of the target gene
directly or may do so by interfering with the function of
an activator protein required for efficient gene
transcription
G8 processing of
eukaryotic pre-mRNA
? Overiew
? 5’ processing capping
? 3’ processing cleavage and polyadenylation
? RNA splicing
? Alternative processing
? RNA editing
5‘Processing capping
? Immediately after transcription,the 5’phosphate is
removed,guanosyltransferase adds a G residue linked via a
5'-5'covalent bond,and this is methylated to form a 7-
methylguneosine cap,
? The ribose residues of either the adjacent one or two
nucleotides may also be methylated by methy1 group
addittn to the 2'OH of the sugar
3'processing,clevage and
polyadenylation
? polyadenylation signal:5'-AAUAAA-3'
? 5'-YA-3'
? A GU-rich sequence
? poly(A) ploymerase,250A residues
3'processing,polyadenylation
3'processing clevage
? The poly(A) tail protects the 3' end of the final mRNA against
nuclease degradation and also increases translational
efficiency of he mRNA,
? Some pre-mRNA are cleaved near the 3'end but no poly(A) tail
is added,
RNA splicing
? The precise removal of intron sequences and joining the
ends of neighboring exons to produce a functional
mRNA molecule,a process called RNA splicing,
RNA splicing
Transesterification reactions
RNA splicing
Alternative processing
? Some pre-mRNAs contain more that one set of sites for
3' end cleavage and polyadenylation,such that the use of
alternative sites can lead to mRNA products that contain
different 3' noncoding regions or have different coding
capacities,
Alternative processing
Alternative splice pathways
? Alternative splice pathways also exist whereby the exons
that are retained in the final mRNA depends upon the
pathway chosen,allowing several different proteins to be
synthesized from a single gene,
? P229.fig G8.8
RNA editing
? RNA editing is the name given to several reactions
whereby the nucleotide sequence on an mRNA
molecule may be changed by mechanisms other than
RNA splicing,
? Individual nucleotides may be substituted,
added(inserted) or deleted,
? P229.fig G8.9
G9 Ribosomal RNA
? Ribosomes
? Transcription and processing of prokaryotic RNA
? Synthesis of eukaryotic 28S,18S and 5.8S rRNA
? Ribozymes
? Synthesis of eukaryotic 5S rRNA
Ribosomes
? Each ribosome consists of two subunits,a small subunit and
a large subunit,each of which is a multicomponent complex
of ribosomal RNAs and ribosomal proteins,
? A sedimentation coefficient沉降系数 (Svedberg
units):size,shape and density of the particle,
Prokaryotic 70S ribosome
Eukaryotic 80S ribosome
E.Coli,ribosome
Trancription and processing of
prokaryotic rRNA
? In E.Coli there are 7 rRNA trancription units scattered
throughout the genome,each of which contains one copy of
each of the 23S,16S and 5S rRNA genes and one to four
copies of various tRNA genes,
Processing of prokaryotic rRNA
Processing of prokaryotic r RNA
Synthesis of eukaryotic28S,18S
and 5.8S rRNA
? In eukaryotic,the gene for 28S,18S and 5.8S rRNA are
typically clustered together and tandemly repeated in
that one copy each of 28S,18S and 5.8S gene
occur,followed by untranscribed Spacer DNA,then
another set of 28S,18S and 5.8S genes occur and so on,
? P234.figG9.4
Synthesis of eukaryotic28S,18S
and 5.8S rRNA
Ribozymes
? In tetrahymena四膜虫,the pre-rRNA molecule contains
an intron that is removed by self-splicing（ in the
presence of guanosine,GTP,GDP,GMP） without the
need for involvement of any protein,
? This was the first ribozyme discovered but many have
since been reported
self-splicing
Self-splicing
Synthesis of eukaryotic 5S rRNA
? RNA Polymerases III
? The promoter contain control elements called the A box
and C box located downstream of the transcriptional
start site,
? P236,
G10 Transfer RNA
? tRNA structure
? Transcription and processing of tRNA in prokaryotes
? Transcription and processing of tRNA in eukaryotes
? Modification of tRNA
tRNA structure
tRNA tertiary structure
tRNA tertiary structure
Transcription and processing of
Prokaryotic tRNA
? E.coli contains clusters of up to seven tRNA genes
separated by spacer regions,as well as tRNA genes
within ribosomal RNA transcript units,
? Following transcription,the pre-RNA transcript folds
up into specific stem-loop structures and is then
processed by ribonucleases D,E,F and P in an
ordered series of reactions to release the individual
tRNA molecules,
Processing of Prokaryotic tRNA
Transcription of tRNA in eukaryotes
? RNA Pol Ⅲ,
? The tRNA promoter includes two control elements,called
the A box and B box,located within the tRNA gene itself
and hence downstream of the transcriptional start site,
? P241.Fig G10.3
Processing of eukaryotic tRNA
Modification of tRNA
? Following synthesis,nucleotides in the rRNA molecule
may undergo modification to create unusual nucleotides
such as 1-methylguanosine (m1G），
pseudouridine(ψ),dihydrouridine(D),inosine(Ⅰ )and 4-
thiouridine(S4U),
Unusual nucleotides
? G2 Transcription in prikaryotes
? G3 The lac operon
? G4 The trp operon
? G5 Transcription in eukaryotes
? G6 Transcription of protein-coding genes
? G7 Regulation of transcription by RNA pol II
? G8 Processing of eukaryotic pre-mRNA
? G9 Ribosomal RNA
? G10 Transfer RNA
G RNA synthesis and processing
Central Dogma
RNA
DNA
Protein
Transcription
Translation
Replication
Reverse
Transcription
Juang RH (2004) BCbasics
Nucleotide
Nucleoside
Phosphoric acid
Pentose
Bases
Bases
Purine
Pyrimidine
A
G
C
U
G1 RNA structure
Nucleic acid
Infrequent bases
? s4U 4-硫尿嘧啶
? Inosine 次黄嘌呤
? m1G 1-甲基鸟嘌呤
? m1A 1-甲基腺嘌呤
? DHU 5,6-二氢尿嘧啶
? s2C 2-硫胞嘧啶
? Pseudouridine(ψ )假尿嘧啶,C1-C5
Rare bases
Nucleoside(C1-N1 or C1-N9)
? Adenosine
? Guanosine
? Cytidine
? Uridine
? Pseudouridine(ψ):C1-C5
Covalent structure
? long polymer chain
? ATP,UTP,GTP,CTP
? 3’-5’ phosphodiester bonds（ not 2’-5’）
? 5’-GUCATUATCCA-3’
Long polymer chain
断裂
RNase
微碱
Difference between RNA and DNA
RNA secondary structure
? RNA molecules are largely single-stranded but there
are regions of self-complementary where the RNA
chain forms internal double-stranded regions
RNA secondary structure
RNA
RNA
tRNA secondary structrue
tRNA
tRNA
? Three phases of transcription
1,Promoters and initiation起始
2,Elongation延伸
3,Termination终止
? RNA processing加工
G2 transcription in prokaryotes
3’ 5’
5’ 3’ RNA
template strand
nontemplate strand (+) strand
(-) strand
5’ 3’
coding strand
AUG
ATG
TAC
UAG
TAG
ATC
Start
codon
Same
sequence
Template strand
Sense(+) strand=coding strand
Antisense(-) strand=template strand
Transcript
5’ 3’
RNA
3’ 5’
DNA
3’ 5’
RNA
5’ 3’
DNA
Promotor
RNA synthesis
RNA polymerase
RNA polymerase
Promoter
? During initiation,RNA polymerases recognizes a
specific site on the DNA,upstream from the gene
that will be transcribed,called a promoter site,
? 启动子是指 RNA 聚合酶识别、结合和开始转录的一
段 DNA序列。
-10sequence:TATAAT(Pribnow box):recognition site
-35sequence:TTGACA is important in DNA unwind
16-18bp
5-8bp
Promoters differ by up to 1000-fold in their
efficiency of initiation of transcription
Promoter
Promoters and initiation
Transcription bubble
Direction of transcription
Elongation
Terminator
Rho depentend terminator
RNA processing
? Induction of the lac operon
? The lac repressor
? CRP/CAP
? Positive and negative regulation
G3 the lac operon
The Lac operon
The lac operon
? The operon is transcribed by RNA polymerase to
produce a single polycistronic mRNA that is then
translated to produce all three enzymes
? These enzymes are involved in lactose metabolism,
? When lactose is absent,E.coli makes only small amounts
of these enzyme,but the presence of lactose induces
synthesis of large amounts of all three enzymes,
3 Structure genes
Lac Z:β-Galactosidase( β-半乳糖苷酶 ),which hydrolyzes
lactose(乳糖 )to glucose(葡萄糖 )and galactose(半乳糖 )
Lac Y:galactoside Permease (β-半乳糖苷透性酶 ),it transports
lactose into the cell across the cell membrane
Lac A:thiogalactosideTransacelylase：硫代 半乳糖苷转乙酰基
酶
Hydrolysis
Induction of the lac operon
? The mechanism of induction is that background level of β
–galactosidase converts some lactose to allolactose异乳
糖 which then acts as an inducer and turns on
transcription of the lac operon,
? IPTG（ isopropylthiogalactoside） can also act as an
inducer,
IPTG
The lac repressor
? Transcription of the operon controlled by the lac
repressor protein encoded by the lac I gene
? The lacI gene has its own promoter to which RNA
polymerase binds and initiates transcription,
The lac repressor
The lac repressor
The inducer
CRP/ACP
? Catabolite activator protein,CAP is an activator required
for high level transcription of the lac operon,
? The active molecule is a CRP dimer that binds 3‘-5'
cAMP to form a CRP-cAMP complex,
? CRP-cAMP binds to the lac promoter and increases the
binding of RNA polymerase,stimulating transcription
of the lac operon,
CRP/ACP
? When glucose is present,the intracellular level of cAMP
falls,CRP cann’t bind to the lac promoter and the lac
operon is only weakly transcribed,
? When glucose is absent,the level of intracellular cAMP
rises,the CRP-cAMP complex stimulates transcription of
the lac operon and allows lactose to be used as an
alternative carbon source,
CRP/ACP
CRP/ACP
Positive and negative regulation
? In negative regulation of prokaryotic gene
expression,bound repressor prevents transcription of the
structural genes,
? In positive regulation of gene expression,an activator
binds to DNA and increnses the rate of transcription,
? Through the lac repressor and CRP/CAP protein,the lac
operon is subject to both negative and positive control,
? Organization of the trp operon
? Repression
? Attenuation
? Attenuation vs,repression
G4 The trp operon
Organization of the trp operon
? The trp operon contains five structural genes encoding
enzymes for tryptophan biosynthesis,a trp promoter and
a trp operator sequence,
The trp operon
? The operon is transcribed only when tryptophan is
scarce,
Repression
? When tryptophan is present,tryptophan synthesis is not
needsd,
? In this situation,acting as a corepressor,
tryptophan binds to the repressor and activates it so that
the repressor now binds to the trp operator and stops
transcription of the trp operon,
The trp operon
Attenuation弱化作用
? The trp operon is also controlled by attenuation,
? A leader sequence in the polycistronic mRNA upstream
of the coding region of the trpE structural gene encodes
a 14 amino acid leader peptide including two tryptophan
residues
Leader sequence
Attenuation
? The RNA leader sequence can for several possible stem-loop
secondary structures,one of which can act as a transcription
terminator whilst a different stem-loop can act as an anti-
terminator,
Attenuation
? In the presence of tryptophan,ribosomes bind to the trp
polycistronic mRNA that is being transcribed,following
closely behind the RNA polymerase and begin to
translate the leader sequence,
? When tryptophan is present,translation of the leader
sequence occurs to completion,
Attenuation of the operon
? If tryptophan is scarce,the ribosome pauses when
attempting to translate the two trp codons in the leader
sequence,which leaves the leader sequence available to
form the anti-terminator stem-loop,
? Transcription of the trp operon is then allowed to continue,
Attenuation of the operon
Attenuation vs repression
? The trp operon is regulated by both repression(which
determines whether transcription will occur or not) and
attenuation (which fine tunes transcription),
? Other operons for amino acid biosynthetic pathways may also
be regulated by both repression and attenuation or only by
attenuation,
? Three RNA polymerases
? RNA synthesis
? RNA polymerases subunits
G5 Transcription in eukaryotes
Three RNA polymerases
? RNA pol I is located in the nucleolus核仁 and transcribes
the 28S,18S and 5.8S rRNA
? RNA pol II is lacated in the nucleoplasm核质 and
transcribes pre-mRNA and snRNAs
? RNA pol III is lacated in the nucleoplasm and transcribes the
genes for tRNA,5S rRNA,U6 snRNA
Three RNA polymerases
RNA synthesis
Precursors:4 dNTPs(N=A,U,G,C) Mg2+
? Each RNA polymerase transcribes only one strand,the
antisense(-)strand of a double – stranded DNA
template,directed by a promoter,
? Synthesis occurs 5' → 3'and not require a primer,
RNA polymerase subunits
? Each of the three RNA polymerases contain 12 or more
subunits,some of which are similar to those of E.coli
RNA polymerases,
? However,four to seven subunits in each enzyme are
unique to that enzyme,
? Gene organization
? Initiation of transcription
? Elongation
? termination
G6 transcription of protein coding
genes in eukaryotes
Gene organization
? Most protein-coding genes in eukaryotes consist of coding
sequences called exons interrupted by noncoding sequences
called introns,
? The number of introns and their size varies from gene to gene,
? The primary transcript undergoes processing reactions to
yield mature mRNA,
mRNA
DNA
5’ 3’
cap
poly A
tail
exon exon exon
intron intron
mature mRNA
Processing
Transcription
Splicing
promotor
3’ 5’ start codon stop codon
to cytoplasm
move intron
3 exons of myoglobin
St
rye
r (
19
95
) B
ioch
em
ist
ry
(4e
) p
.15
3
Mathews et al (2000) Biochemistry (3e) p.231
Initiation of transcription
? Most promoter sites for RNA polymerase II have a TATA
box located about 25 bp upstream of the transcriptional
start site,
TATA box
Transcription factors
? RNA polymerase binding to the promoter requires the
formation of a transcription initiation complex involving
several general transcription factors that assemble in a
strict order,
Elongation and termination
? Elongation continues until transcription comes to a halt
at varying distances downstream of the gene,releasing
the primary RNA transcript,pre- mRNA,
? This molecule then undergoes processing reactions to
yield mRNA,
G7 regulation of transcription
by RNA pol II
? Mechanism of regulation
? Upstream of regulation
? Enhancers
? Transcription factors have multiple domains
? DNA binding domains
? Dimerization domains
? Activation domains
? repressors
Mechanism of regulation
? Transcriptional regulation occurs via transcription factors
that bind to short control elements associated with the
target genes and then interact with each other and with
the transcription initiation complex to increase or
decrease the rate of transcription of the target gene,
NOTES
Transcription factors：与转录控制有关的蛋白质
Cis-acting elements(顺式作用元件 ),转录因子识别并结合的
DNA调控区。
Tans-acting factors(反式作用元件 )：与顺式元件结合的蛋白
质转录因子。
Enhancers(增强子 )：促进启动子的转录活性的一类顺式元件
Upstream regulatory elements
? Many transcription factors bind to control elements
located upstream within a few hundred base-pairs of the
protein-coding gene,
? The SP1 box and CAAT box are examples of such
regulatory elements found upstream of most protein-
coding genes,but some upstream regulatory elements
are associated with only a few genes and are
responsible for gene-specific transcriptional regulation,
The SP1 box and CAAT box
Enhancers
? positive transcriptional control elements
? 100-200 bp
? upstream or downstream,
? active in a long distance away,
? The transcription factors bound to these long-distance
elements interact with the transcription initiation complex
by looping out of the DNA,
Enhancers
Transcription factors have
multiple domains
? A DNA-binding domain that recognizes the specific DNA
control element to bind to
? An activation domain that interacts with other
transcription factors or the RNA polymerases,
Helix-turn-helix
Helix-turn-helix
Helix-turn-helix
Zinc finger
Zinc finger
Zinc finger
A leucine zippers
Leu zipper
A leucine zippers
Helix-loop-helix motif
Helix-loop-helix motif
Activation domains
? No common structural motifs are known for the
activation domains of transcription factors,
1,Acid Activation domains,rich in acidic amino acids
2,Glutamine-rich domains
3,Proline-rich domains
Repressors
? Repressor proteins that inhibit the transcription of
specific genes in eukaryotes may bind either to control
elements near to the target gene or to silencers that may
be located a long distance near to the target gene,
? The repressor may inhibit transcription of the target gene
directly or may do so by interfering with the function of
an activator protein required for efficient gene
transcription
G8 processing of
eukaryotic pre-mRNA
? Overiew
? 5’ processing capping
? 3’ processing cleavage and polyadenylation
? RNA splicing
? Alternative processing
? RNA editing
5‘Processing capping
? Immediately after transcription,the 5’phosphate is
removed,guanosyltransferase adds a G residue linked via a
5'-5'covalent bond,and this is methylated to form a 7-
methylguneosine cap,
? The ribose residues of either the adjacent one or two
nucleotides may also be methylated by methy1 group
addittn to the 2'OH of the sugar
3'processing,clevage and
polyadenylation
? polyadenylation signal:5'-AAUAAA-3'
? 5'-YA-3'
? A GU-rich sequence
? poly(A) ploymerase,250A residues
3'processing,polyadenylation
3'processing clevage
? The poly(A) tail protects the 3' end of the final mRNA against
nuclease degradation and also increases translational
efficiency of he mRNA,
? Some pre-mRNA are cleaved near the 3'end but no poly(A) tail
is added,
RNA splicing
? The precise removal of intron sequences and joining the
ends of neighboring exons to produce a functional
mRNA molecule,a process called RNA splicing,
RNA splicing
Transesterification reactions
RNA splicing
Alternative processing
? Some pre-mRNAs contain more that one set of sites for
3' end cleavage and polyadenylation,such that the use of
alternative sites can lead to mRNA products that contain
different 3' noncoding regions or have different coding
capacities,
Alternative processing
Alternative splice pathways
? Alternative splice pathways also exist whereby the exons
that are retained in the final mRNA depends upon the
pathway chosen,allowing several different proteins to be
synthesized from a single gene,
? P229.fig G8.8
RNA editing
? RNA editing is the name given to several reactions
whereby the nucleotide sequence on an mRNA
molecule may be changed by mechanisms other than
RNA splicing,
? Individual nucleotides may be substituted,
added(inserted) or deleted,
? P229.fig G8.9
G9 Ribosomal RNA
? Ribosomes
? Transcription and processing of prokaryotic RNA
? Synthesis of eukaryotic 28S,18S and 5.8S rRNA
? Ribozymes
? Synthesis of eukaryotic 5S rRNA
Ribosomes
? Each ribosome consists of two subunits,a small subunit and
a large subunit,each of which is a multicomponent complex
of ribosomal RNAs and ribosomal proteins,
? A sedimentation coefficient沉降系数 (Svedberg
units):size,shape and density of the particle,
Prokaryotic 70S ribosome
Eukaryotic 80S ribosome
E.Coli,ribosome
Trancription and processing of
prokaryotic rRNA
? In E.Coli there are 7 rRNA trancription units scattered
throughout the genome,each of which contains one copy of
each of the 23S,16S and 5S rRNA genes and one to four
copies of various tRNA genes,
Processing of prokaryotic rRNA
Processing of prokaryotic r RNA
Synthesis of eukaryotic28S,18S
and 5.8S rRNA
? In eukaryotic,the gene for 28S,18S and 5.8S rRNA are
typically clustered together and tandemly repeated in
that one copy each of 28S,18S and 5.8S gene
occur,followed by untranscribed Spacer DNA,then
another set of 28S,18S and 5.8S genes occur and so on,
? P234.figG9.4
Synthesis of eukaryotic28S,18S
and 5.8S rRNA
Ribozymes
? In tetrahymena四膜虫,the pre-rRNA molecule contains
an intron that is removed by self-splicing（ in the
presence of guanosine,GTP,GDP,GMP） without the
need for involvement of any protein,
? This was the first ribozyme discovered but many have
since been reported
self-splicing
Self-splicing
Synthesis of eukaryotic 5S rRNA
? RNA Polymerases III
? The promoter contain control elements called the A box
and C box located downstream of the transcriptional
start site,
? P236,
G10 Transfer RNA
? tRNA structure
? Transcription and processing of tRNA in prokaryotes
? Transcription and processing of tRNA in eukaryotes
? Modification of tRNA
tRNA structure
tRNA tertiary structure
tRNA tertiary structure
Transcription and processing of
Prokaryotic tRNA
? E.coli contains clusters of up to seven tRNA genes
separated by spacer regions,as well as tRNA genes
within ribosomal RNA transcript units,
? Following transcription,the pre-RNA transcript folds
up into specific stem-loop structures and is then
processed by ribonucleases D,E,F and P in an
ordered series of reactions to release the individual
tRNA molecules,
Processing of Prokaryotic tRNA
Transcription of tRNA in eukaryotes
? RNA Pol Ⅲ,
? The tRNA promoter includes two control elements,called
the A box and B box,located within the tRNA gene itself
and hence downstream of the transcriptional start site,
? P241.Fig G10.3
Processing of eukaryotic tRNA
Modification of tRNA
? Following synthesis,nucleotides in the rRNA molecule
may undergo modification to create unusual nucleotides
such as 1-methylguanosine (m1G），
pseudouridine(ψ),dihydrouridine(D),inosine(Ⅰ )and 4-
thiouridine(S4U),
Unusual nucleotides
