transcription: rna synthesis, rna processing, “algorithms ...transcription start, termination, rna...
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Biology introduction
“Algorithms for genomes”2b
Dr. Jan M. Kooter
Department of Genetics, FALW
jan.kooter@falw.vu.nl
http://www.bio.vu.nl/genetica/
Transcription: RNA synthesis, RNA processing,Intron-exon structure
Promoters: Regulation, Chromatin structure, CpGislands
Protein domains: Modular structure of proteins,exon-shuffling
Protein-homologs: paralogues and orthologues
Central Dogma
RNA
promoter
Transcription start and termination
Start Stop
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RNA Polymerases Where does RNA pol bind?
Pribnow Box of Promoter: ProkaryoteTemplate (Anticoding) Strand
>> terminologie
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Initiation ofTranscription(prokaryote)
- RNA pol.- Sigma factor
-
Terminator Regions of DNA
- Rho-independent vanwege de U’s!(Rho-dependent terminators donot have these Us
Transcription Overview: prokaryoteDNA Transcribed into RNA
Some terminology: 5’ en 3’ Untranslated region(leader, trailer)
‘upstream’‘downstream’
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In prokaryotes: Transcription and Translationare coupled!
Not the case in eukaryotes: transport of mRNAfrom nucleus to cytoplasm
Transcription start, termination, RNA processing in eukaryotes
(A)n
DNA(gene)
pre-mRNA
mRNA
transcription
Splicing (Spliceosome)
Protein
translation
promoter
Important difference between pro- and eukaryotes isthat genes in eukaryotes consist of exons and introns.
Cap
Cap
RNA Polymerase IITranscription-initiationcomplex
(many subunits !!)
RNA Polymerase II: promoterRegulation
Interactions between proteins bound to promoter elements
Specific sequences recognized by“transcription factors” (activators,repressors)
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Example: Yeast Transcriptional Factor:DNA binding: “cis-acting elements”
DNA(promoter element=cis-acting element)
Transcription factor
DNA is associated/packaged with proteins:Chromatin
DNA winds aroundhistone proteins (nucleosomes).
Other proteins wind DNA into more tightlypacked form, thechromosome.
Unwinding portions of the chromosome isimportant for mitosis,replication and makingRNA.
Histone tails extend beyond the nucleosome, andare sites of (mostly) reversible post-translational
modification
H3 and H4 have been most extensively studied to date
Basically: The type of histone modification (mostly acetylation andmethylation) and the position of the modified amino acid determineswhether a gene will be expressed or not. Transcription factors andassociated proteins can modifiy the amino acids in the histone tails.
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Ac
Ac
Ac
Ac
Ac
HAT
• The action of Histone acetyl transferases (HATs) both decondenseschromatin, and provides recognition sites for bromodomain containingproteins (eg. Gcn5)
Bromodomain
CpG islands:- CpG rich regions (GC% >50%; >200 bp long, often much longer>1000 bp, observed/expected ratio of CpG > 0.6)- Part of a promoter, and actually function as promoter- Current estimate > 50% of the genes in human genomecontain a CpG island- CpG are unmethylated- But, when methylated, it leads to transcriptional repression- Several tumors contained methylated CpG islands !!!
An example of a CpG Island in the Retinoblastoma gene region. Thedotted line represents the statistically expected frequency of CpG sites(1/16), while the solid line represents the measured frequency of CpGsites in the 180 kb of DNA sequence that encompass the Rb gene exonsand introns. The location of two CpG islands is indicated by arrows. Onlythe most 5' CpG island corresponds to the promoter of the gene.
Example of a CpG island in the Retinoblastoma gene
http://www.mdanderson.org/departments/methylation/
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Transcription start, termination, RNA processing in eukaryotes
(A)n
DNA(gene)
pre-mRNA
mRNA
transcription
Splicing (Spliceosome)
Protein
translation
promoter
- Important difference between pro- and eukaryotes is thatgenes in eukaryotes consist of exons and introns.- Introns removed by a process called “splicing”
Cap
Cap
- capping (7m-Guanosine addition at 5’ of mRNA)
- poly-adenylation (50-200 Adenosines at 3’ of mRNA)
-splicing (removal of intron(s)): nuclear process>>> mRNA in cytoplasma does not contain introns
>> Transport of mRNA from nucleus to cytoplasm:Active and regulated process.
Processing of pre-mRNAs RNA polymerase II synthetised RNAs:
Messenger RNA 5' Cap Example:Mouse
Beta-GlobinGene
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Nuclear Introns and splicing(removal of intron sequences)
- Exon-intron junctions characterized by specific bases sequences Conservation, although there are junctions with different bases!
>> Property can be used to identify genes and exon/introns
Nuclear mRNA Intron RemovalRol van U-RNAs: herkenning van exon-intron grenzen en “branch-
point”
Intron Removal in Nuclear RNA“Splicing” Gene recognition in the genome
- Scan genomic DNA for exon/intron sequences, promotersequences, open reading frames, etc.
-Relevant informations comes from RNA because genesequences are expressed via RNA
ÿ Copy DNA sequences ( isolate mRNA > cDNA > sequence) - compare cDNA (no introns!) wih genomic DNA
ÿ Expressed sequence tags (ESTs, not necessarily completemRNAs, non-coding RNAs) (RNAs > cDNA) - compare ESTs with genomic DNA
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Protein domains and exon-shufflinghypothesis for the assembly and origin of
“new” genes.
Many Proteins have a modular structure: functional domains> Each domain has a specific function, and can be shared bydifferent proteins: Some proteins contain multiple copies ofa domain.
Examples:
Modular build-up of proteins: visualized Model: Assembly of different modules into a single proteinoccurs via exon shuffling at the DNA level:
>> 1 or more exons encode for a particular protein domain; By DNArearrangements or via a RNA, exon sequences can be duplicated andinserted in other genomic sites; for example, in other genes.With this mechanism, it is assumed that new genes are created.
Not on scale: Principle!
Calmodulin: Ca-binding Kinase (activated by Calmodulin- Ca+ )
Model: by exon-shuffling DNAs combined
2 distinctproteins
Both functions intoone protein
Example
Senses [Ca++ ]
- Calmodulin and kinase (enzyme that phosphorylates proteins), are often separate één eiwit. Calcium bindend eiwitdomein en kinase domein veelal gecodeerd door aparte eiwitten: In Arabidopsis en veel andere planten speciefieke domeinen samengebracht.
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Principle exon-shuffling: (Know principle and how to apply in order to explain modular build-up of genes/proteins)
Protein / gene homology
Definition of important concepts in (comparative) genomics
- Orthologous genes or orthologs:
Genes are orthologous if their divergence reflects a speciation event.
Have similar developmental and physiological functions and very similar
in protein sequence
Example: alpha-globin (human) < - > alpha-globin (chimpanzee)
- Parologous genes or paralogs:
Genes are paralogous if their divergence reflects a gene duplication event
within a species, and have novel functions.
Example: alpha-globin (human) < - > beta-globin (human) < - > myoglobin (human)
- Homologous genes or homologs:
Genes derived from a common ancestral gene. Level of similarity often reflects
the time they diverged:
>>>> Homologs can be devided in orthologs and paralogs
Gene/Protein Evolution
• Homologs– Common ancestor
– Common 3D Structure
– At least some sequence similarity(sequence motifs or more closesimilarity)
• > Paralog– Derived by duplication
• > Ortholog– Derived by Speciation
Anastasia Nikolskaya, GeorgetownUniv.
Homologs > paralogs + orthologs
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Orthologs and Paralogs
Myx
inid
ae
Tele
osto
mi
Verteb
rata
Tetrap
oda
Mam
malia
Amph
ibia
Hb
(Hag
fish
)
Myo
(C
od)
HbA
(C
od)
HbB
(C
od)
Myo
(Fr
og)
HbA
(Fr
og)
HbB
(Fr
og)
Myo
(R
at)
HbA
(R
at)
HbB
(R
at)
Myo
(H
agfi
sh)
CraniataAnastasia Nikolskaya, Georgetown Univ
Thanks for your attention !!
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