l1 rna-syntes och processning_ht11_bilda
DESCRIPTION
RNA-synthesis, RNA-Processing, Central DogmaTRANSCRIPT
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1L1: RNA Synthesis and Processing
Outline
Evolution of the gene concept
Transcription in Prokaryotes
Eukaryotic RNA Polymerases and General
Transcription Factors
Regulation of Transcription in Eukaryotes
RNA Processing and Turnover
Gene expression
Changes in gene expression, rather than losses of genetic
material, play a key role in guiding and maintaining cell
differentiation.
Gene expression changes regulate cell behaviours
including cell fate decisions during embryogenesis, cell
function, and chemotaxis, and can even lead to changes
in an entire organism, such as molting in insects
Gene expression is regulated by cell-extrinsic and cell-
intrinsic factors
What was a gene in da old days?
1860s-1900s: a discrete unit of heredity
1910s: a distinct locus
1940s: a blueprint for a protein
1960s: a transcribed code
1970s-1980s: an open reading frame (ORF) pattern
What was a gene yesterday (1990s-2000s)?
A DNA segment that contributes to phenotype/function. Inthe absence of demonstrated function a gene may becharacterized by sequencee, transcription or homology Wain et al, Genomics, 2002
A locatable region of genomic sequence, which isassociated with regulatory regions, transcribed regionsand/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 theGene Sweepstake team, 2003
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2The encode project-home work!
Nature 2007 Jun 14;447(7146):799-816
Some ENCODE findings
The human genome is pervasively transcribed, such that themajority of bases are associated with at least one primarytranscript, and many transcripts link distal regions toestablished protein coding loci.
Many novel non-protein coding transcripts have been identified,many of which overlap protein-coding loci and others locatedin regions previously thought to be silent
Numerous previously unrecognized transcript start sites havebeen identified.
Biological complexity revealed by ENCODE
Gerstein M B et al. Genome Res. 2007;17:669-6812007 by Cold Spring Harbor Laboratory Press
What is a gene today (post ENCODE project)?
A gene is a genomic sequence (DNA or RNA) directly encodingfunctional product molecules, either RNA or protein
In the case that there are several functional products sharingoverlapping regions, one takes the union of all overlappinggenomic sequences coding for them
This union must be coherent i.e., done separately for finalprotein and RNA products but does not require that allproducts necessarily share a common subsequence
or just
The gene is a union of genomic sequences encoding acoherent set of potentially overlapping functionalproducts
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3How the proposed definition of the gene can be applied to a sample case
Gerstein M B et al. Genome Res. 2007;17:669-681
2007 by Cold Spring Harbor Laboratory Press
The encode project-home work!
Genome Res. 2007 17: 669-681
What is a gene, post-ENCODE?History and updated definitionMark B. Gerstein, Can Bruce, Joel S. Rozowsky, Deyou Zheng, Jiang Du,Jan O. Korbel, Olof Emanuelsson, Zhengdong D. Zhang, Sherman Weissman,and Michael Snyder
While sequencing of the human genome surprised us with how many protein-coding genes there are, it did notfundamentally change our perspective on what a gene is. In contrast, the complex patterns of dispersed regulationand pervasive transcription uncovered by the ENCODE project, together with non-genic conservation and theabundance of noncoding RNA genes, have challenged the notion of the gene. To illustrate this, we review theevolution of operational definitions of a gene over the past centuryfrom the abstract elements of heredity ofMendel and Morgan to the present-day ORFs enumerated in the sequence databanks. We then summarize thecurrent ENCODE findings and provide a computational metaphor for the complexity. Finally, we propose a tentativeupdate to the definition of a gene: A gene is a union of genomic sequences encoding a coherent set of potentiallyoverlapping functional products. Our definition sidesteps the complexities of regulation and transcription byremoving the former altogether from the definition and arguing that final, functional gene products (rather thanintermediate transcripts) should be used to group together entities associated with a single gene. It also manifestshow integral the concept of biological function is in defining genes.
What is a gene?
gene
structural gene
RBS
promotorers
transcription start ATGGTG
TTG
TAA
TAG
TGA
signal peptide
transcription unit
transcription termination signal
Transcription in prokaryotes
1. Initiation: RNA-polymerase binds to the
promoter, conformational changes in the
promoter-polymerase complex (a bubble), initial
transcription
2. Elongation: conformational changes (tightened
grip around the template), unwinding of DNA,
RNA-synthesis, proof-reading
3. Termination: Rho-independent terminators
(intrinsic terminators = hairpin structures) or Rho-
dependent terminators
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4E. coli RNA polymerase
RNA polymerase: 2+ (ca 465 kDa)
- sigma factor influences the DNA binding
properties of the polymerase
- the affinity to different promoters can differ by
a factor of 106
- transcription is initiated w/o primer
Sequences of E. coli promoters
16-19 bp 5-9 bp
Start signals for RNA synthesis
Strong promoter: high affinity to RNA polymerase
Constitutive promoter: always on
Inducible promoter: possible to turn on and off at will
Sigma factors () in E. coli
stress responseSrpoS
heat shockErpoE
GCCGATAA15 bpCTAAAmotility/chemotaxis28 (F)fliA
TTGCA6 bpCTGGNAN-metabolism54rpoN
CCCGATNT13-15 bpCCCTTGAAheat shock32rpoH
TATAAT16-19 bpTTGACAgeneral70rpoD
-10sep-35UseFactorGene
Transcription by E. coli RNA polymerase (Part 1)
~ 12-14
bp
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5Transcription by E. coli RNA polymerase (Part 2)
(after ca 10 bp)
Transcription termination in Prokaryotes
RNA synthesis continues until the polymeraseencounters a termination signal.
The most common signal is a symmetricalinverted repeat of a GC-rich sequence followedby seven A residues.
Alternatively, transcription of some genes isterminated by a specific termination protein(Rho), which binds extended segments ofsingle-stranded RNA.
Transcription termination Transcription termination (E. coli)
5
5
DNA
RNA
Promoter RNA polymerase
Hairpin termination signal
Other mechanism:
Termination via Rho protein (hexamer)
Rho
Pause-site
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6Transcription regulation
Two modes: positive and negative regulation
Positive regulation: transcription factors (activator/s) must bind
to the promoter in order for transcription to be initiated
- they help the RNA polymerase to bind the DNA (recruitment)
- allosteric influence on processes after the polymerase has
bound
Negative regulation: repressor protein binds to the operator
and prevents transcription
- hinders the RNA polymerase from binding the promoter
- alternatively, the RNA polymerase is retained at the promoter
Gene regulation after transcription initiation
Premature transcription termination, attenuation; e.g., trp
Anti termination = a type of positive transcription regulation in
which trancription factors (proteins) bind RNA polymerase and
modifies it so that it can read through special termination sites
- used by phages and in certain operons
Metabolism of lactose Negative control of the lac operon
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7Positive control of the lac operon by glucose Regulation of the lactose operon (E. coli)
Lac Z Lac Y Lac A
Lac Z Lac Y Lac AS.D. S.D. S.D.
5 3
mRNA
DNA
-galactosidase:
Turns lactose into
galactose and glucose
Lactose permease:
Regulates the lactose
uptake
Thio-galactoside-acetylase:
Degrades non-cleavable
lactose analogues
mRNA
5 3Lac I
Lac I-35 -10-35 -10
Catabolite Activator
Protein (CAP)
Cyclic AMP
(cAMP)
cAMP/CAP-binding
region
mRNA-start
In the absence of lactose: Lac-repressor binds to the operator sequence Transcriptionen is blocked; no -galactosidase (or any of the other enzymers) is producced
Lactose (or IPTG) present: Lac-repressor cannot bind to the operator sequence
Transcription is allowed; -galactosidase (and all the other enzymes) is produced
Presence of lactose (or IPTG) and low concentrations of glucose: cAMP concentration rises
cAMP-CAP-complex formed that can bind upstream of the promoter
cAMP-CAP-complex promotes transcription (guides the RNA polymerase)
More -galactosidase is produced
Operator-sequence
Lac-repressor(Lac I)
Lac I
CAP
Promoter=Landing spot for
RNA polymerase
Lactose orIPTG
(synthetic analogue)
S.D.
RNA-pol.
Promoter
Eukaryotic RNA Polymerases and General Transcription Factors
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
RNA polymerase II is responsible for synthesis of mRNA and ithas been the focus of most transcription studies.
Unlike prokaryotic RNA polymerase, it requires initiation factorsthat (in contrast to bacterial factors) are not associated withthe polymerase.
General transcription factors are proteins involved intranscription from all polymerase II promoters.
About 10% of the genes in the human genome encodetranscription factors, emphasizing the importance of theseproteins.
Promoters contain several different regulatory sequenceelements.
Promoters of different genes contain different combinations ofpromoter elements, which appear to function together to bindgeneral transcription factors.
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8Transcription Factors & Transcription Control in Eukaryotic Cells
How did eukaryotic organisms become so much morecomplex than prokaryotic ones, without a whole lotmore genes?
The answer lies in transcription factors (TFs)!
TFs recognizes specific DNA sequences and usually havemultiple functional domains for binding to e.g. TFs,coactivators, RNA pol II, chromatin remodelingcomplexes, ncRNAs etc.
The complexity and fine gradation of DNA expression ineukaryotes result from combinatorics; the combination ofchromatin and TF signals rather than the individual TFsignal is read out.
Transcription Factors & Transcription Control in Eukaryotic Cells
Does more cellular complexity require more genes?
Not really! E.g., it is estimated that humans haveapproximately one billion different kinds of neurons in thebrain while C. elegans (a roundworm) only has a total of 302neurons despite having nearly as many genes as we do.
The key to cell differentiation lies in the combination oftranscription factors (TFs) and chromatin structuresduring specific points of transition
Formation of a polymerase II preinitiation complex in vitro (Part 1) Formation of a polymerase II preinitiation complex in vitro (Part 2)
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9RNA 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
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A eukaryotic promoter The SV40 enhancer
Action of enhancers DNA looping
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The immunoglobulin enhancer Structure of transcriptional activators
Examples of DNA-binding domains Action of transcriptional activators
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Action of eukaryotic repressors Regulation of transcriptional elongation (Part 1)
Regulation of transcriptional elongation (Part 2) Regulation of transcriptional elongation (Part 3)
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Regulation of Transcription in Eukaryotes
The packaging of eukaryoticDNA in chromatin hasimportant consequences fortranscription, so chromatinstructure is a critical aspect ofgene expression.
Modifications of chromatinstructure play key roles in thecontrol of transcription ineukaryotic cells (thought tohelp lock in expressionchanges needed duringdevelopment).
Actively transcribed genes arein relatively decondensedchromatin, which can be seenin polytene chromosomes ofDrosophila.
Regulation of Transcription in Eukaryotes
Chromatin can be modified by:
Interactions with HMG (high mobility
group) proteins
Modifications of histones
Rearrangements of nucleosomes
The modifications have either silencing or promoting
effects on gene expression
Histone acetylation (Part 1)
Histone modification:
The amino-terminal tail domains of corehistones are rich in lysine and can bemodified by acetylation (promotestranscription).
Transcriptional activators and repressorsare associated with histoneacetyltransferases (HAT) anddeacetylases (HDAC), respectively.
Patterns of histone modification
Can increase or decrase histone acetylation, thus having positive
and negative effetct on transcription, respectively
Promotes
transcription
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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 (gene silencing).
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.
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Processing of ribosomal RNAs Processing of transfer RNAs (Part 1)
Processing of transfer RNAs (Part 2) Processing of eukaryotic messenger RNAs (Part 1)
(7-methylguanosine)
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Processing of eukaryotic messenger RNAs (Part 2) Formation of the 3 ends of eukaryotic mRNAs
RNA Processing and Turnover: Splicing
Three sequence elements of pre-mRNAs areimportant:- 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 ateach of these positions.
Splicing takes place in large complexes, calledspliceosomes, which have five types of smallnuclear RNAs (snRNAs)U1, U2, U4, U5, and U6.
They are complexed with six to ten protein moleculesto form small nuclear ribonucleoprotein particles(snRNPs).
Splicing of pre-mRNA
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Alternative splicing in Drosophila sex determination Alternative splicing of Dscam
12x48x33x2=38016 combinations!!
RNA Processing and Turnover: RNA editing
RNA editing is processing (other than splicing) thatalters the protein-coding sequences of somemRNAs.
It involves single base modification reactions suchas deamination of cytosine to uridine andadenosine to inosine.
Editing of the mRNA for apolipoprotein B, whichtransports lipids in the blood, results in twodifferent proteins:
Apo-B100 is synthesized in the liver bytranslation of the unedited mRNA.
Apo-B48 is synthesized in the intestine fromedited mRNA in which a C has been changedto a U by deamination.
Editing of apolipoprotein B mRNA
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RNA Processing and Turnover: RNA degradation
Aberrant mRNAs can also be degraded.
Nonsense-mediated mRNA decay eliminatesmRNAs that lack complete open-reading frames.
When ribosomes encounter premature terminationcodons, translation stops and the defective mRNAis degraded.
Ultimately, RNAs are degraded in the cytoplasm.
Intracellular levels of any RNA are determined by abalance between synthesis and degradation.
Rate of degradation can thus control geneexpression.
RNA Processing and Turnover
rRNAs and tRNAs are very stable, in bothprokaryotes and eukaryotes.
Bacterial mRNAs are rapidly degraded, mosthave half-lives of 2 to 3 minutes.
In eukaryotic cells, mRNA half-lives vary; lessthan 30 minutes to 20 hours in mammalian cells.
Short-lived mRNAs code for regulatoryproteins, levels of which can vary rapidly inresponse to environmental stimuli.
mRNAs encoding structural proteins or centralmetabolic enzymes have long half-lives.
RNA Processing and Turnover
Degradation of eukaryote mRNAs is initiated byshortening of the poly-A tails.
Rapidly degraded mRNAs often contain specificAU-rich sequences near the 3 ends which arebinding sites for proteins that can eitherstabilize them or target them for degradation.
These RNA-binding proteins are regulated byextracellular signals, such as growth factors andhormones.
Degradation of some mRNAs is regulated by bothsiRNAs and miRNA.
mRNA degradation
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Summary
Transcription in prokaryotes and eukaryotes hassimilarities but is much more complex in eukaryotes
Many modes for regulation:- specificity factors- repressors/activators- general TFs- enhancers- chromatin structure- DNA methylation- post-transcriptional regulation
RNA processing is very important
Synthesis and turnover determines the final RNA levels;turnover is an important control mechanism
Identification of TFs & regulatory elements-home work!
Learn more about the following commonmethods for identification of TFs & regulatoryelements (use textbooks and/or internetbased info)
Transcription factor binding: DNA footprinting (Part 1) Transcription factor binding: DNA footprinting (Part 2)
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Transcription factor binding: Electrophoretic-mobility shift assay Transcription factor binding:
DNA footprinting
Gel shift assay
Identification of eukaryotic regulatory sequences Transcription factor binding: Chromatin immunoprecipitation (I)
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Transcription factor binding: Chromatin immunoprecipitation (II)