L1: RNA Synthesis and Processing
Outline
L1: RNA Synthesis and
Processing
• Transcription in Prokaryotes
• Eukaryotic RNA Polymerases and
General Transcription Factors
• Regulation of Transcription in
Eukaryotes
• RNA Processing and Turnover
Vad är en gen?
What was a gene yesterday?
gen
promotorer
signalpeptid
transkriptionsstart
RBS
transkriptionstermineringssignal
TAA
TAG
TGA
ATG
GTG
TTG
strukturell gen
transkriptionsenhet
What is a gene today?
“A DNA segment that contributes to phenotype/function. In
the absence of demonstrated function a gene may be
characterized by sequencee, transcription or homology” –
Wain et al, Genomics, 2002
“A locatable region of genomic sequence, which is
associated with regulatory regions, transcribed regions
and/or other functional sequence regions” – Pearson,
Nature, 2006
“Alternatively spliced transcripts all belong to the same gene,
even if the proteins that are produced are different” – the
Gene Sweepstake team, 2003
The encode project-home work!
A gene is a genomic sequence (DNA or RNA) directly encoding
functional product molecules, either RNA or protein
In the case that there are several functional products sharing
overlapping regions, one takes the union of all overlapping
genomic sequences coding for them
This union must be coherent – i.e., done separately for final
protein and RNA products – but does not require that all
products necessarily share a common subsequence
or just
The gene is a union of genomic sequences encoding a coherent
set of potentially overlapping functional products
Nature 2007 Jun 14;447(7146):799-816
1
E. coli RNA polymerase
Transkription i prokaryoter
1. Initiering: RNA-polymeras binder promotor,
strukturella förändringar hos promotorpolymeraskomplexet (”bubbla”), initial
transkription
2. Elongering: konformationsförändringar (håller
hårdare om templatet), uppvindning av DNAt,
RNA-syntes, ”proof-reading”
3. Terminering: Rho-oberoende terminatorer
(inneboende terminatorer = hårnålsstrukturer),
Rho-beroende terminatorer
DNA footprinting (Part 1)
Sequences of E. coli promoters
16-19 bp
RNA polymeras: α2ββ´ω+ σ (ca 465 kDa)
- sigmafaktorn påverkar DNA-bindande
egenskaperna hos polymeraset
- bindningsstyrkan till olika promotorer kan
skilja 106 x
- transkription initieras utan primer
5-9 bp
• Startsignaler för RNA-syntes
• Stark promotor: hög affinitet för RNA-polymeras
• Konstitutionell promotor: alltid påslagen
• Reglerbar promotor: går att slå på och stänga av
DNA footprinting (Part 2)
Sigmafaktorer (σ) i E. coli
Gene Factor
Use
-35
sep
-10
rpoD
σ70
general
TTGACA
16-19 bp
TATAAT
rpoH
σ32
heat shock
CCCTTGAA
13-15 bp
CCCGATNT
rpoN
σ54
N-metabolism
CTGGNA
6 bp
TTGCA
fliA
σ28 (σF )
motility/chemotaxis
CTAAA
15 bp
GCCGATAA
rpoE
σE
heat shock
rpoS
σS
stress response
2
Transcription by E. coli RNA polymerase (Part 1)
Transcription by E. coli RNA polymerase (Part 2)
•
•
Structure of bacterial RNA polymerase
(after ca 10
bp)
~ 12-14
bp
Transcription termination in Prokaryotes
RNA synthesis continues until the polymerase
encounters a termination signal.
The most common signal is a symmetrical
inverted repeat of a GC-rich sequence followed
by seven A residues.
Alternatively, transcription of some genes is
terminated by a specific termination protein
(Rho), which binds extended segments of
single-stranded RNA.
Transcription termination
Transcription termination (E. coli)
Promoter
RNA polymerase
DNA
RNA
”Hairpin”
termination signal
5´
5´
Other mechanism:
Termination via
Rho protein (hexamer)
Pause-site
Rho
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Transkriptionsreglering
Genreglering efter transkriptionsinitiering
• Prematur transkriptionsterminering s.k. attenuering; ex. trp
• Skiljer på positiv och negativ reglering
• Positiv reglering: transkriptionsfaktor (aktivator) måste binda in
till promotorn för att transkription skall ske
- hjälper RNA-polymeras att binda till DNAt (rekrytering)
- allosterisk verkan av steg efter polymerasinbindning
• Antiterminering = en typ av positiv transkriptionsreglering vid
vilken transkripionsfaktorer (proteiner) binder till RNApolymeras och modifierar det så att det kan läsa igenom
speciella termineringssites
- används av fager och i vissa operon
• Negativ reglering:repressorprotein binder till operator och
hindrar transkription
- hindrar RNA-polymeras att binda till promotorn
- alternativt håller kvar RNA-polymeraset
Metabolism of lactose
Negative control of the lac operon
Positive control of the lac operon by glucose
Reglering av laktos-operonet (E. coli)
Catabolite
Activator
Protein
(CAP)
Promotor
-35
Cykliskt
Promotor=
AMP ”Landningsplats” för
(cAMP)
RNA-polymeras
CAP
-10
Lac I
S.D.
5´
Lac-repressor
(Lac I)
Laktos eller
IPTG
(syntetisk analog)
DNA
-10
Lac Z
Lac I
mRNA
Lac I
mRNA-start
RNA-pol.
-35
3´
cAMP/CAPbindningsställe
Operatorsekvens
S.D.
5´
Lac Z
β-galaktosidas:
Spjälkar laktos till
galaktos och glukos
Lac Y
S.D.
Lac Y
Laktos-permeas:
Reglerar införsel
av laktos
Lac A
S.D.
mRNA
Lac A
3´
Thio-galaktosid-acetylas:
Bryter ned
ej klyvbara laktos-analoger
Frånvaro av laktos:
• Lac-repressorn binder till operator-sekvensen
• Transkriptionen blockeras; inget β-galaktosidas-enzym (eller något av de andra enzymerna) produceras
Närvaro av laktos (eller IPTG):
• Lac-repressorn kan inte binda till operator-sekvensen
• Transkriptionsker; β-galaktosidas-enzym (och alla de andra enzymerna) produceras
Närvaro av laktos (eller IPTG) samt låga halter av glukos:
• Halten av cAMP stiger
• cAMP-CAP-komplex bildas som kan binda uppströms om promotorn
• cAMP-CAP-komplex främjar transkriptionen (vägleder RNA-polymeraset)
• Mer β-galaktosidas-enzym produceras
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Eukaryotic RNA Polymerases and General Transcription Factors
Structure of yeast RNA polymerase II
Eukaryotic cells have three nuclear RNA
polymerases that transcribe different
classes of genes.
They are complex enzymes, consisting of
12 to 17 different subunits each.
They all have 9 conserved subunits, 5 of
which are related to subunits of bacterial
RNA polymerase.
Eukaryotic RNA Polymerases and General Transcription Factors
Formation of a polymerase II preinitiation complex in vitro (Part 1)
RNA polymerase II is responsible for synthesis of mRNA and it
has been the focus of most transcription studies.
Unlike prokaryotic RNA polymerase, it requires initiation factors
that (in contrast to bacterial σ factors) are not associated with
the polymerase.
General transcription factors are proteins involved in
transcription from all polymerase II promoters.
About 10% of the genes in the human genome encode
transcription factors, emphasizing the importance of these
proteins.
Promoters contain several different regulatory sequence
elements.
Promoters of different genes contain different combinations of
promoter elements, which appear to function together to bind
general transcription factors.
Formation of a polymerase II preinitiation complex in vitro (Part 2)
Model of the polymerase II preinitiation complex
5
RNA polymerase II/Mediator complexes and transcription initiation
The ribosomal RNA gene is transcribed by RNA polymerase I
Initiation of rDNA transcription
Transcription of RNA polymerase III genes
Identification of eukaryotic regulatory sequences
A eukaryotic promoter
6
The SV40 enhancer
Action of enhancers
DNA looping
The immunoglobulin enhancer
Electrophoretic-mobility shift assay
Chromatin immunoprecipitation (Part 1)
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Chromatin immunoprecipitation (Part 2)
Structure of transcriptional activators
Examples of DNA-binding domains
Action of transcriptional activators
Action of eukaryotic repressors
Regulation of transcriptional elongation (Part 1)
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Regulation of transcriptional elongation (Part 2)
Regulation of transcriptional elongation (Part 3)
Regulation of Transcription in Eukaryotes
Regulation of Transcription in Eukaryotes
The packaging of eukaryotic
DNA in chromatin has
important consequences for
transcription, so chromatin
structure is a critical aspect of
gene expression.
Chromatin can be modified by:
Modifications of chromatin
structure play key roles in the
control of transcription in
eukaryotic cells.
• Modifications of histones
• Interactions with HMG (high mobility
group) proteins
• Rearrangements of nucleosomes
Actively transcribed genes are
in relatively decondensed
chromatin, which can be seen
in polytene chromosomes of
Drosophila.
Histone acetylation (Part 1)
Patterns of histone modification
•
Histone modification:
•
The amino-terminal tail domains of core
histones are rich in lysine and can be
modified by acetylation.
Transcriptional activators and repressors
are associated with histone
acetyltransferases (HAT) and
deacetylases (HDAC), respectively.
•
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Chromatin remodeling factors
Chromatin remodeling factors are protein complexes that alter contacts between DNA
and histones.
They can reposition nucleosomes, change the conformation of nucleosomes, or eject
nucleosomes from the DNA.
Regulation of Transcription in Eukaryotes
To facilitate elongation, elongation factors become
associated with the phosphorylated C-terminal domain of
RNA polymerase II.
They include histone modifying enzymes and chromatin
remodeling factors that transiently displace nucleosomes
during transcription.
Regulation of Transcription in Eukaryotes: DNA methylation
•
•
DNA methylation is another general mechanism that controls
transcription in eukaryotes.
Methyl groups are added at the 5-carbon position of cytosines (C)
that precede guanines (G) (CpG dinucleotides).
RNA Processing and Turnover
Most newly-synthesized RNAs must be modified, except
bacterial RNAs which are used immediately for protein
synthesis while still being transcribed.
rRNAs and tRNAs must be processed in both prokaryotic
and eukaryotic cells.
Regulation of processing steps provides another level of
control of gene expression.
Processing of ribosomal RNAs
Processing of transfer RNAs (Part 1)
10
Processing of transfer RNAs (Part 2)
Processing of eukaryotic messenger RNAs (Part 1)
Processing of eukaryotic messenger RNAs (Part 2)
Formation of the 3´ ends of eukaryotic mRNAs
Splicing of pre-mRNA
RNA Processing and Turnover: Splicing
Three sequence elements of pre-mRNAs are
important:
Sequences at the 5′ splice site, at the 3′ splice site,
and within the intron at the branch point.
Pre-mRNAs contain similar consensus sequences at
each of these positions.
Splicing takes place in large complexes, called
spliceosomes, which have five types of small
nuclear RNAs (snRNAs)—U1, U2, U4, U5, and U6.
They are complexed with six to ten protein molecules
to form small nuclear ribonucleoprotein particles
(snRNPs).
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Alternative splicing in Drosophila sex determination
Alternative splicing of Dscam
12x48x33x2=38016 combinations
RNA Processing and Turnover: RNA editing
Editing of apolipoprotein B mRNA
RNA editing is processing (other than splicing) that
alters the protein-coding sequences of some
mRNAs.
It involves single base modification reactions such
as deamination of cytosine to uridine and
adenosine to inosine.
Editing of the mRNA for apolipoprotein B, which
transports lipids in the blood, results in two
different proteins:
Apo-B100 is synthesized in the liver by
translation of the unedited mRNA.
Apo-B48 is synthesized in the intestine from
edited mRNA in which a C has been changed
to a U by deamination.
RNA Processing and Turnover: RNA degradation
Aberrant mRNAs can also be degraded.
Nonsense-mediated mRNA decay eliminates
mRNAs that lack complete open-reading frames.
When ribosomes encounter premature termination
codons, translation stops and the defective mRNA
is degraded.
Ultimately, RNAs are degraded in the cytoplasm.
Intracellular levels of any RNA are determined by a
balance between synthesis and degradation.
Rate of degradation can thus control gene
expression.
RNA Processing and Turnover
rRNAs and tRNAs are very stable, in both
prokaryotes and eukaryotes.
Bacterial mRNAs are rapidly degraded, most have
half-lives of 2 to 3 minutes.
In eukaryotic cells, mRNA half-lives vary; less than
30 minutes to 20 hours in mammalian cells.
Short-lived mRNAs code for regulatory proteins,
levels of which can vary rapidly in response to
environmental stimuli.
mRNAs encoding structural proteins or central
metabolic enzymes have long half-lives.
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RNA Processing and Turnover
mRNA degradation
Degradation of eukaryote mRNAs is initiated by
shortening of the poly-A tails.
Rapidly degraded mRNAs often contain specific AUrich sequences near the 3′ ends which are binding
sites for proteins that can either stabilize them or
target them for degradation.
These RNA-binding proteins are regulated by
extracellular signals, such as growth factors and
hormones.
Degradation of some mRNAs is regulated by both
siRNAs and miRNA.
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