Download Chapter 11 Transcription and RNA Processing

Chapter 11
Transcription and RNA Processing
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Chapter Outline
Transfer of Genetic Information: The Central
Dogma
The Process of Gene Expression
Transcription in Prokaryotes
Transcription and RNA Processing in
Eukaryotes
Interrupted Genes in Eukaryotes: Exons and
Introns
Removal of Intron Sequences by RNA
Splicing
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Transfer of Genetic Information:
The Central Dogma
Transfer of Genetic Information:The Central Dogma
The central dogma of biology is
that information stored in DNA
is transferred to RNA molecules
during transcription and to
proteins during translation.
DNA
RNA
Genotyping
RNA
proteins
Phenotyping
DNA/RNA
virus
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proteins
The Central Dogma
Transcription
--one strand of DNA to one
complementary strand of RNA
--Uracil replaces Thymine
(U-A bp)
--primary transcripts
messenger RNA (mRNA)
-----chemical modification
(splice=remove introns)
(spliceosomes)
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The Central Dogma
Translation
--RNA nucleotide into amino acids
UUU--Phe ( F) phenylalanine(*)
--genetics code (codons)
--Ribosomes (ribonucleoproteins)
(*) sense codon
Non sense codon=stop codon
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Transcription and Translation
in Prokaryotes
 The primary transcript is
equivalent to the mRNA
molecule.
 The mRNA codons on the
mRNA are translated into an
amino acid sequence by the
ribosomes.
 Retroviruses
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Transcription and Translation
in Eukaryotes
 The primary transcript (premRNA) is a precursor to the
mRNA.
 The pre-mRNA is modified
at both ends, and introns are
removed to produce the
mRNA.
3
 After processing, the mRNA
is exported to the cytoplasm
for translation by ribosomes.
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Types of RNA Molecules
 Messenger RNAs (mRNAs)—intermediates that carry genetic
information from DNA to the ribosomes.
 Transfer RNAs (tRNAs)—adaptors between amino acids and
the codons in mRNA.
 Ribosomal RNAs (rRNAs)—structural and catalytic
components of ribosomes.
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Types of RNA Molecules
 Small nuclear RNAs (snRNAs)—s tructural components of
spliceosomes.
 Micro RNAs (miRNAs)—short single-stranded RNAs (20 to 22
bp) that block expression of complementary mRNAs.
 RNAi is similar to miRNA (RNA interference, double strand
RNA, plant) siRNA (small interference)
 piRNA (Piwi-interacting RNA) is a small non-coding RNA
molecules
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The Central Dogma
• The central dogma of molecular biology is that genetic
information flows
-----from DNA to DNA during chromosome replication,
----from DNA to RNA during transcription,
----from RNA to protein during translation.
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 Transcription involves the synthesis of a single strand RNA
transcript complementary to one strand of DNA of a gene.
• Translation is the conversion of information stored in the
sequence of nucleotides in the RNA transcript into the
sequence of amino acids in the polypeptide gene product,
according to the specifications of the genetic code.
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The Process of Gene Expression
The Process of Gene Expression
For non-viral proteins…
Information stored in the nucleotide sequences of
genes is translated into the amino acid
sequences of proteins through unstable
intermediaries called messenger (m)RNAs.
Synthesis of viral proteins… in infected
bacteria involved an unstable RNA molecule
synthesized from the viral DNA.
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RNA synthesis
1-Single strand of DNA
(template strand)
2-ribonuleoside triphosphate
(NTP)
3-no pre-existing primers (de
novo)
template strand=transcribed
Protein
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Nucleophilic attacks
--3’ hydroxyl group of the RNA
strand
--nucleotidyl phosphorus on the
nucleoside triphosphate
“nucleotide,
nucleoside monophosphate”
“RNA polymerase”
“Transcriptional factors”
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The Transcription Bubble
Prokaryotes:
--RNA polymerase binds specific
nucleotide sequences (promoter
regions) plus transcriptional
factors
--Single RNA polymerase
--DNA unwinding (AT regions)
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The Transcription Bubble
Eukaryotes:
--several RNA polymerases
--no direct recognition binding
--transcriptional factors
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General Features of RNA Synthesis
 Similar to DNA Synthesis except
– The precursors are ribonucleoside triphosphates.
– Only one strand of DNA is used as a template.
– RNA chains can be initiated de novo (no primer required).
 The RNA molecule will be complementary to the DNA template
(antisense) strand and identical (except that uridine replaces
thymidine) to the DNA non-template (sense) strand.
 RNA synthesis is catalyzed by RNA polymerases and proceeds
in the 5’3’ direction. © John Wiley & Sons, Inc.
• In eukaryotes, genes are present in the nucleus, whereas
polypeptides are synthesized in the cytoplasm.
• Messenger RNA molecules function as non-stable
intermediaries that carry genetic information from DNA to the
ribosomes, where proteins are synthesized.
• RNA synthesis, catalyzed by RNA polymerases, is similar to
DNA synthesis in many respects.
• RNA synthesis occurs within a localized region of strand
separation (Transcription Bubble), and only one strand of
DNA functions as a template for RNA synthesis.
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• RNA synthesis, catalyzed by RNA polymerases, is
similar to DNA synthesis in many respects.
Prokaryotic:
OriC (245 bp)
AT-rich region (replication bubble)
Eukaryotic:
ARS (Autonomously Replicating Sequences)
AT-rich region 11 bp
T
O
T
cDNA
5’
3’
5’
3’
http://www.youtube.com/watch?v=mFh9L-nu8Hk
http://www.youtube.com/watch?v=OnuspQG0Jd0
Transcription in Prokaryotes
Transcription
---The first step in gene expression
---Transfers the genetic information stored in
DNA (genes) into messenger (m)RNA molecules
that
---Carry the information to the sites of protein
synthesis in the cytoplasm.
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Stages of Transcription
--DNA dependent RNA polymerase
--5’ to 3’ direction
--Walk (literally) on the DNA
--Upstream and downstream
regions
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E. Coli RNA Polymerase
Tetrameric core: 2 ’
Holoenzyme: 2 ’ 
 (480,000 Daltons; bp~650 Daltons)
Functions of the subunits:




: assembly of the tetrameric core
: ribonucleoside triphosphate binding site
’: DNA template binding region
(sigma factor): initiation of transcription (*)
(*) in vivo
In vitro: RNA polymerase works…just fine on both DNA strands
Initiation of RNA Chains

Binding of RNA polymerase holoenzyme to a
promoter region in DNA ( promoter region)

Localized unwinding of the two strands of DNA by
RNA polymerase to provide a single-stranded
template

Formation of phosphodiester bonds between the
first few ribonucleotides in the nascent RNA chain
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A Typical E. coli Promoter
..,-2,-1,+1,+2,..
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Numbering of a Transcription Unit
 The transcript initiation site is +1 (A/T).
 Bases preceding the initiation site are given minus (–)
prefixes and are referred to as upstream
sequences.
 Bases following the initiation site are given plus (+)
prefixes and are referred to as downstream
sequences.
 Consensus sequences: highly conserved
 Recognition sequences: Sigma factor (
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Elongation
Sigma factor needs to be released
---Re- and Un-winding activities
-- Walk (literally) on the DNA
5’ to 3’
--growing RNA chain
RNA polymerase binds both
DNA template and growing RNA chain
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Termination Signals in E. coli
Rho-dependent terminators—require a
protein factor ()
Rho-independent terminators—do not
require 
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Rho-independent terminators—do not require 
intrinsic termination)
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RNA transcription stops
--when the newly synthesized RNA
molecule forms a G-C-rich hairpin loop
followed by a run of As
--Create a mechanical stress
--Pulls the poly-U transcript out of the
active site of the RNA polymerase
--A-U has very weak interaction
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Termination Signals in E. coli
Rho-dependent terminators (non-intrinsic) —
require a protein factor () and rut site
Rut proteins bind specific RNA sequences
(>>Cs and <<<Gs)
Not hairpins or other secondary Structures
Rho utilization (rut)
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QuickTime™ and a
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QuickTime™ and a
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Coupled Transcription and
Translation in E. coli
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QuickTime™ and a
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QuickTime™ and a
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stop (aka "nonsense") codon
QuickTime™ and a
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Rho factor binds to specific regions in naked RNA
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• RNA synthesis occurs in three stages: (1) initiation, (2)
elongation, and (3) termination.
• RNA polymerases—the enzymes that catalyze
transcription—are complex multimeric proteins.
• The covalent extension of RNA chains occurs within
locally unwound segments of DNA.
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• Chain elongation stops when RNA polymerase
encounters a transcription-termination signal.
• Transcription, translocation, and degradation of mRNA
molecules often occur simultaneously in prokaryotes.
RNAase
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Transcription and RNA Processing in Eukaryotes
Five different enzymes catalyze transcription in
eukaryotes, and the resulting RNA transcripts undergo
three important modifications, including the excision of
noncoding sequences called introns.
The nucleotide sequenced of some RNA transcripts are
modified post-transcriptionally by RNA editing.
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Modifications to Eukaryotic pre-mRNAs
 A 7-Methyl guanosine cap is added to the 5’ end of
the primary transcript by a 5’-5’ phosphate linkage.
( stability and protection)
 A poly(A) tail (a 20-200 nucleotide polyadenosine
tract, As) is added to the 3’ end of the transcript. The
3’ end is generated by cleavage rather than by
termination. (stability and protection)
 When present, intron sequences are spliced out of
the transcript. (stability)
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Eukaryotes Have Five RNA Polymerases
RNA polymerase II Nucleus
miRNA
Pre-mRNA~Heterogeneous nuclear RNA (hnRNA)
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A Typical RNA Polymerase II Promoter (mRNA)
Promoter: short sequence of conserved elements (seq. of DNA)
located upstream from the transcript starting point.
--~200 bp ( DNA linear)
--~10 Kdp ( DNA bending)
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Initiation by RNA Polymerase II
Transcriptional factors (proteins)
--Help/modulate/assist
Basal transcriptional factors
--bind close to the transcript starting point
Other factors (enhancers and silencers)
-TFIID
--TATA-biding proteins (TBP)
-TFIIA
-TFIIB
Where is the enzyme?
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-TFIIF (together with the RNA Pol-II)
--enzymatic activity (DNA-unwinding)
-TFIIE
--binds downstream regions
-TFIIH (helicase activity)
-TFIIJ
--binds downstream regions
Helicase activity:
it separates two annealed nucleic acid strands
RNA Pol-II DNA-unwinding activity:
RNA Polymerase bends and wraps around DNA.
TFIIF alters (nonspecific) DNA binding by RNA polymerase II,
resulting in substantial DNA unwinding but not DNA strand separation.
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-TFIIH (helicase activity and kinase activity)
When RNA polymerase II binds to
the complex, it initiates transcription.
Phosphorylation of the CTD is
required for elongation to begin.
CTD: carboxy-terminal domain
 All eukaryotic RNA polymerases have ∼12 subunits and are aggregates of
>500 kD. (nucleotide pair~0.660 kD)
 Some subunits are common to all three RNA polymerases.
 The largest subunit in RNA polymerase II has a CTD (carboxy-terminal
domain) consisting of multiple repeats of a heptamer.
-Typical RNA polymerase isolated from yeast (S.
cerevisiase) ( and  subunits)
-  subunits: CTD – carboxy-terminal domain, which
consists in multiple repeats of 7 amino acids, unique
and important of regulation (tyrosine (Try, Y), serine
(Ser, S) and threonine (Thr, T) residues)
-Some subunits are common to all three polymerases.
Figure 24.2
RNA Polymerase I Has a Bipartite Promoter
 The RNA polymerase I promoter consists of:
 --a core promoter
 --an upstream control element (UPE)
RNA Pol I transcribes rRNA genes.
Core promoter: -45 to +20 seq.,
G-C-rich and A-T-rich (Inr-initiator) regions,
Binding factors - protein complexes formed by
TFIs and TBP-(TATA binding protein)
RNA Polymerase III Uses Both
Downstream and Upstream Promoters
RNA polymerase III has (3) types of promoters.
-RNA Pol III transcribes tRNA
-Core promoters (boxes)
-Transcriptional Factors(TF) III:
general and specifics
*proximal sequence element
*
Figure 24.7
RNA Chain elongation
--Model
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The 7-Methyl Guanosine
(7-MG) Cap
Histones:?
FACT (facilitates chromatin transcriptional)
Energy
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RNA Chain termination
The 3’ Poly(A) Tail
Termination signal: specific DNA seq.
-1000 to 2000 nucleotides
--AAUAAA seq.
--GU-rich seq.
Endonuclease
--poly(A) polymerase
Pol-II vs Pol I and III
-Terminator proteins (Rho-indep.
Terminator)
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RNA Editing
Usually the genetic information is not altered in
the mRNA intermediary.
Sometimes RNA editing changes the
information content of genes by
– Inserting or deleting uridine monophosphate
residues.
– Changing the structures of individual bases
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Editing of Apoplipoprotein-B mRNA
(Amino groups)
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•
Three to five different RNA polymerases are
present in eukaryotes, and each polymerase
transcribes a distinct set of genes.
•
Eukaryotic gene transcripts usually undergo three
major modifications:
•
•
•
the addition of 7-methyl guanosine caps to 5’ termini,
The addition of poly(A) tails to 3’ ends,
The information content of some eukaryotic transcripts is
altered by RNA editing, which changes the nucleotide
sequences of transcripts prior to their translation.
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Interrupted Genes in Eukaryotes:
Exons and Introns
Most eukaryotic genes contain
noncoding sequences called introns
that interrupt the coding sequences, or
exons.
The introns are excised from the RNA
transcripts prior to their transport to the
cytoplasm.
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Hybridization: annealing
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R-Loop Evidence of an Intron in the
Mouse -Globin Gene
mRNA
Missing in
actions
Pre-mRNA
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Introns
 Introns (or intervening sequences) are noncoding sequences
located between coding sequences.
 Introns are removed from the pre-mRNA and are not present in
the mRNA.
 Introns are variable in size and may be very large ( 50 bp to 3000
bp).
 Exons (both coding and noncoding sequences) are composed of
the sequences that remain in the mature mRNA after splicing.
 The biological significance of introns is still open to debate.
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Removal of Intron Sequences
by RNA Splicing
The noncoding introns are
excised from gene transcripts
by several different
mechanisms.
Eukaryotes
No prokaryotes (excepts a few a prokaryotes virus and others)
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Excision of Intron Sequences
Conserved seq. for mRNA
Exon-GT…AG-exon
intron
99%
Ribonucleoproteins:
Spliceosomes (1981)
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Splicing
Removal of introns must be very precise.
Conserved sequences for removal of the introns of
nuclear mRNA genes are minimal.
– Dinucleotide sequences at the 5’ and 3’ ends of introns.
Exon-GU…AG-exon
– TACTAAC box (branch site with A) about 30 nucleotides
upstream from the 3’ splice site.
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Spliceosomes:
snRNA plus ~40 proteins
1%: CG…AG
AT…AC
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Nuclear splicing
involves transesterification
GU…UACUAAC….AG
“Branch site”
Does splicing require energy?
Does splicing ever occur in
DNA?
Figure 26.7
tRNA
 A splicing endonuclease makes two cuts at the end
of the intron.
 A splicing ligase joins the two ends of the tRNA to
produce the mature tRNA.
 Specificity resides in the three-dimensional
(secundary) structure of the tRNA precursor, not in
the nucleotide sequence.
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rRNA
(Autocatalytic Splicing)
G-3’-OH:
absolute requirement
“Co-factor”
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• Noncoding intron sequences are excised
from RNA transcripts in the nucleus prior to
the transport to the cytoplasm.
• Introns in tRNA precursors are removed by
the concerted action of a splicing
endonuclease and ligase, whereas introns in
some rRNA precursors are spliced out
autocatalytically—with no catalytic protein
involved.
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• The introns in nuclear pre-mRNAs are
excised on complex ribonucleoprotein
structures called spliceosomes.
• The intron excision process must be precise,
with accuracy to the nucleotide level, to
ensure that codons in exons distal to
introns are read correctly during translation.
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