Chapter 11. RNA synthesis and processing (P169, sP841)

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" The Central Dogma of molecular biology"
replication
transcription
Reverse transcription
translation
Chapter 10
Transcription
(RNA Biosynthesis)
Transcription*:
RNA biosynthesis from a DNA template
is called transcription.
transcription
DNA
•products:mRNA tRNA rRNA
RNA
Enzymes and Proteins involved in transcription :
•substrates :
NTP
(
GTP, CTP
ATP,
)
•template:
DNA
• enzyme :
RNA polymerase
• the other Protein factors
UTP,
Chemical reaction-- polymerization reaction:
RNA polymerase catalyze formation of Phosphodiester
bonds and release pyrophosphate (ppi)
RNA polymerase
RNA precursor
RNA biosynthesis is similar to DNA biosynthesis*:
Template- DNA
Enzyme—dependent on DNA
Chemical reaction--the formation of
Phosphodiester bonds
Direction of synthesis--- 5’
 obey the ruler of base paired
3’
RNA biosynthesis includes three stages:
Initiation: RNA polymerase binds to the
promoter of DNA, and then a
transcription “bubble” is formed.
Elongation: the polymerase catalyzes
formation of 3’5’-phosphodiester bonds
in 5’3’ direction, using NTP as building
units.
Termination: when the polymerase
reaches a termination sequence on DNA,
the reaction stops and the newly
synthesized RNA is released.
Formation of a transcription bubble
1. RNA biosynthesis in prokaryotes
RNA polymerase in E. coli :
consists of five subunits, a2bb’ws, which is called
“holoenzyme”. The s subunit functions as a starting
factor that can recognize and bind to the promoter
site.
The rest of the enzyme, a2bb’w, is known as “core
enzyme”, responsible for elongation of the RNA
sequence.
E. coli RNA polymerase
b
b
b
b
a
a
Core enzyme
a
s
a
a
Holoenzyme
1) Important terms in RNA biosynthesis.
A)Operon*: a coordinated unit of gene expression,
which usually contains a regulator gene and a set
of structural genes.
B) Promoter site*: a region of DNA templates that
specifically binds RNA polymerase and determines
where transcription begins.
regulator gene
5
3
Promoter site
structural genes
RNA-pol
3
5
The –10 sequence: refers to the consensus
TATAAT, and is known as “Pribnow box”.
The –35 sequence: refers to the consensus
TTGACA, which is recognized by the s
subunit of RNA polymerase,
DNA template
-35
TTGACA
-10
TATAAT
Pribnow box
recognition site
+1
RNA
5
3
-5 0
-40
-30
-20
-10
1
10
3
5
consensus
-35
sequences
region
the site of transcription
(the start site)
-10
region
TTGACA
AA C T G T
recognition site
T A T A A T Pu
A T A T T A Py
(Pribnow box)
C) Sense and antisense strand:
The antisense (-) strand refers to the DNA strand
that is used as template to synthesize mRNA.
The sense (+) strand of a DNA double helix is the
non-template strand that has the same sequence as that
of the RNA transcript except for T in place of U.
Antisense (-) strand = template strand
Sense (+) strand = coding strand
Sense and antisense strand:
coding strand
antisense strand
5
3
3
5
template strand
sense strand
3) Process of RNA biosynthesis: The process is
similar to DNA synthesis but no primer is
needed and T is replaced by U.
A) Initiation:
•σ factor recognizes the initiation site(-35 region), the
holoenzyme of RNA-pol bind to duplex DNA and move
along the double helix towards –10 region.
•the holoenzyme of RNA-pol arrived on –10 region,and
bind to –10 region ,DNA is partially unwound and was
opened 10-20 bp length.
•Then incoming 2 neighbour nucleotides which base
pairs are complementary with DNA template, RNA
polymerase catalyzed the first polymerization reaction.
5’-pppG -OH + NTP
– 5’ -pppGpN – OH + ppi
s
pppG NTP- OH
pppGpN
ppi
initiation complex:
RNApol(α2ββˊσ)-DNA-pppGpN-OH3’
DNA template
TTGACA
TATAAT
DNA template
TTGACA
TATAAT
DNA template
TTGACA
TATAAT
DNA template
TTGACA
TATAAT
+
“Core”
s
The first phosphodiester bond formed
B) Elongation: after the first phosphodiester
bond has been formed, the s subunit is released.
The core enzyme moves in a 5’3’ direction
on the DNA strand while it is catalyzing
elongation of the RNA transcript.
DNA template
TTGACA
TATAAT
DNA template
TTGACA
TATAAT
NTP
DNA template
TTGACA
TATAAT
tanscription complex:
RNA-pol (core enzyme) ···· DNA ···· RNA
RNA polymerase
Sense strand
Direction of
transcription
Rewinding
3’
5’
5’
3’
5’PPP
Unwinding
Antisense strand
Newly synthesized
RNA strand
C) Termination: when the core enzyme
reaches a termination sequence, the region
near the 3’end of RNA forms a hairpin
structure by self base-pairing. The
transcription stops, the core enzyme and the
newly synthesized RNA are released.
For those DNA templates that lack the
sequence to produce a hairpin structure of
the RNA transcript, a protein factor called
“r ” recognizes the termination site, stops
transcription, and causes release of the newly
synthesized RNA.
A hairpin structure at the 3’end of RNA
C
U
U
G
G
G•C
A•U
C•G
C•G
G•C
C•G
C•G
G•C
5’
A-U-U-U-U-OH 3’
Termination by hairpin structure of RNA
RNA-polymerase
5
3
5’pppG
3
5
DNA
5
RNA
3
RNA polymerase
Ribosome
The multiple-site transcription in bacteria
Subunits of RNA polymerase in E. coli
Subunit Size (AA)
Function
a
329
required for assembly of the
enzyme; interacts with some
regulatory proteins; involved
in catalysis
b
1342
involved in catalysis: chain
initiation and elongation
b'
1407
binds to the DNA template
s
613
directs the enzyme to the
promoter
w
91
required to restore denatured
RNA polymerase in vitro to
its fully functional form
4) Post-transcriptional modification:
The newly synthesized precursors of rRNA
and tRNA in bacteria undergo a series of
process.
A) Processing of rRNA: the 16S, 23S, and 5S
rRNAs in prokaryotes are produced by
cleavage of a rRNA precursor, catalyzed by
ribonuclease III. Additional processes
include methylation of bases and sugar
moieties of some nucleotides.
Processing of rRNAs
16S
23S
5S
First cleavage
16S
23S
5S
Second cleavage
16S(1.5kb)
23S(2.9kb)
5S(0.12kb)
B) Processing of tRNA:
The removal of the 5’ end of tRNA
precursors is catalyzed by RNase P. RNase
P is a ribozyme consisting of RNA that
possesses enzyme activity.
Other processes include the addition of
nucleotides (CCA) to the 3’-end of tRNA,
and formation of some unusual residues
such as pseudo-U, I, T, methyl-G, and DHU,
etc.
Modification of
some residues in
tRNAs
4) Inhibition of transcription:
Rifampicin: an antibiotic that specifically
inhibits the initiation of transcription by
blocking the formation of the first several
phosphodiester bonds in RNA biosynthesis.
Streptolydigin: binds to bacterial RNA
polymerase and inhibits elongation of RNA
chain.
Actinomycin D: binds to DNA and prevents
transcription (at low concentrations it
doesn't affect DNA replication)
2. RNA biosynthesis in eukaryotes
1) RNA polymerases in eukaryotes: three
enzymes, each of which contains 12 or more
subunits.
Polymerase
Pol I
Pol II
Pol III
location
nucleolus
nucleoplasm
nucleoplasm
RNAs transcribed
28S, 18S, 5.8S rRNA
pre-mRNA, snRNA
tRNA, 5S rRNA, U6
snRNA, 7S RNA
2) Process of eukaryotic RNA synthesis
A) Initiation: similar to Pribnow box, a start
site consensus (called TATA box) at –25 is
required for the recognition by RNA
polymerase in eukaryotes.
A A
TATA A
T T
-25
Structural gene
+1
Pol II requires several transcription factors
to start transcription:
TFII-A: to stabilize the TFIID-TATA box complex;
TFII-B: to link Pol II to the initiation complex;
TFII-D: to recognize and bind to the TATA box;
TFII-E: to interact with Pol II and TFII-B;
TFII-F: to form Pol II-TFIIF complex. It also has
DNA helicase activity;
TFII-H, -J: to form the initiation complex.
TATA box
Structural gene
S
TFII-D TBP
S
TBP
A
B
S
A TBP
B
Pol II F
F
S
A TBP
B
E
A
S
TBP
FE
H
B
J
H
J
B) Elongation : after the initiation complex
has formed, the RNA polymerase catalyzes
transcription in a 5’3’direction, using the
(-) DNA strand as template.
Soon after the 5’end of the extending RNA
chain appears from the polymerase complex,
a cap structure is added at the end.
Cap structure of mRNA
O
CH3
N+
N
N
HN
H2N
O
-
O
P
O CH2 O
O
-
O
H
P
O
H
O H
O
-
7-methylguanylate
H
OH
P
OH
O
O
CH2 O
H
Base
H
H
H
O
-
O
P
OCH3
O
O
CH2 O
H
H
Base
H
H
O
OCH3
C) Termination: Two mechanisms may cause
termination of RNA transcription:
A hairpin structure formed at the 3’end of the
nascent RNA causes stop of transcription, as is
seen in the prokaryotic RNA synthesis.
A stop signal sequence, AAUAAA, near the
3’end results in the recognition and binding by
a specific endonuclease, which cleaves the
nascent RNA chain and stops transcription.
The newly synthesized mRNA precursor is
then added a poly A tail by poly A polymerase.
Cleavage and polyadenylation of a mRNA precursor
RNA polymerase
Template DNA
AAUAAA
Nascent RNA
Cleavage signal
endonuclease
ATP
Poly A polymerase
PPi
5’
AAUAAA
mRNA precursor
AAAA(A)n-OH 3’
3) Processing of eukaryotic RNA precursors:
A) Gene organization: protein-coding genes
in eukaryotic DNA are organized in a
discontinuous fashion. The protein-coding
sections are called “exons”, which are
interrupted by noncoding sections called
“introns”.
Exon 1
Promoter
Transcription
initiation site
Exon 1
Intron 1
Exon 1
Intron 2
Transcription
Termination region
B) RNA splicing: a process in which introns of a premRNA are removed to produce a functional
mRNA.
Exon 1
Promoter
Exon 2
Intron 1
Exon 3
Intron 2
Transcription
Exon 1
Exon 2
Exon 3
5’
AA(A)250 3’
Intron 1
Intron 2
RNA splicing
5’
AA(A)250 3’
C) Steps in RNA splicing: usually the exonintron boundaries are marked by specific
sequences. The intron starts with GU and
ends with AG.
Intron
3’ splice site
5’ splice site
Exon 1 GU
CURAY
Branch point
sequence
U/C11
Polypyrimidine
tract
AG Exon 2
I.
II.
Formation of a lariat intermediate: the
phosphodiester bond of the 5’ splice site
is attacked by the 2’-OH of the residue A
in the branch point, forming a 2’5’bond
and releasing the exon 1 with a new 3’OH end.
Connection of exons: The new 3’-OH
end attacks the phosphodiester bond at
the 3’splice site causing the two exons to
join and releasing the intron.
RNA splicing requires the small nuclear
ribonucleoprotein particles (snRNP), each
of which consists of a small nuclear RNA
and several proteins. They are named U1,
U2, U3….
snRNPs bind to the pre-mRNA to form a
complex, called spliceosome, which brings
the two neighbored exons together for
splicing.
Exon 1 GU
A
AG Exon 2
snRNPs
U1 U2
U1
U2
Exon 1 GU
A
AG Exon 2
U4-U5-U6
U1
Exon 1 GU
U5
U2 A
U6 U4
AG Exon 2
Spliceosome
U1
Exon 1 GU
U5
U2 A
U6 U4
AG Exon 2
U1
Exon 1
GU
U5
U2 A
U6 U4
AG Exon 2
Lariat intermediate
U1
Exon 1 Exon 2
GU
U5
U2 A
U6 U4
AG-OH 3’
4) Alternative processing:
A) Alternative polyadenylation sites: this will
cause different splice-sites and produce
different mRNAs with varied lifetimes.
Poly A
Exon 1
Exon 2
Splicing
Poly A
Exon 3
B) Alternative splicing: will cause different
combinations of exons from a primary
transcript of a single gene. This may be
resulted from regulatory proteins that
control the use of certain splice-sites.
Exon 1
Exon 2
Splicing
Exon 3
5) RNA editing: refers to the reactions that
can change the nucleotide sequence of an
mRNA molecule by non-splicing
mechanisms. The change may include:
nucleotide(s) change, deletion, and
insertion.
e.g. the mRNA for apolipoprotein B in the
liver is translated to apolipoprotein B100,
while in the small intestine the mRNA is
changed to yield a new termination codon
(UAA), resulting in a much shorter protein,
apolipoprotein B48.
Apolipoprotein B mRNA
CAA
n
Editing (deamination)
Tr
an
s
lat
io
NH4
UAA
Edited mRNA
translation
ApoB100
ApoB48
Lipoprotein
assembly
LDL receptor
binding
Lipoprotein
assembly
3. Reverse transcription and RNA
replication
1) Reverse transcription: biosynthesis of
DNA using RNA as a template.
 It is important for some viral infections.
These viruses are called retroviruses, such
as some tumor viruses and HIV.
 Reverse transcription is also a powerful
tool in molecular biological techniques or
genetic engineering, such as RT-PCR.
2) RNA replication:
 RNA replication occurs in some viruses.
These viruses encode RNA-directed RNA
polymerase that catalyzes biosynthesis of
RNA from an RNA template.
 RNA replication helps the RNA viruses
easily reproduce their progeny viruses.
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