Download “Algorithms for genomes” 2b Central Dogma Transcription start and

“Algorithms for genomes”
2b
Biology introduction
Transcription: RNA synthesis, RNA processing,
Intron-exon structure
Promoters: Regulation, Chromatin structure, CpG
islands
Protein domains: Modular structure of proteins,
exon-shuffling
Dr. Jan M. Kooter
Department of Genetics, FALW
[email protected]
Protein-homologs: paralogues and orthologues
http://www.bio.vu.nl/genetica/
Central Dogma
Transcription start and termination
promoter
Start
Stop
RNA
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RNA Polymerases
Pribnow Box of Promoter: Prokaryote
Where does RNA pol bind?
Template (Anticoding) Strand
>> terminologie
2
Terminator Regions of DNA
Initiation of
Transcription
(prokaryote)
- RNA pol.
- Sigma factor
- Rho-independent vanwege de U’s!
(Rho-dependent terminators do
not have these Us
Transcription Overview: prokaryote
DNA Transcribed into RNA
Some terminology: 5’ en 3’ Untranslated region
(leader, trailer)
‘upstream’
‘downstream’
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In prokaryotes: Transcription and Translation
are coupled!
Not the case in eukaryotes: transport of mRNA
from nucleus to cytoplasm
Transcription start, termination, RNA processing
in eukaryotes
DNA(gene)
promoter
pre-mRNA
Cap
transcription
Splicing (Spliceosome)
mRNA
Cap
(A)n
translation
Protein
Important difference between pro- and eukaryotes is
that genes in eukaryotes consist of exons and introns.
RNA Polymerase II
Transcription-initiationcomplex
(many subunits !!)
RNA Polymerase II: promoter
Regulation
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 is associated/packaged with proteins:
Chromatin
DNA winds around
histone proteins
(nucleosomes).
Transcription factor
DNA
(promoter element=
cis-acting element)
Histone tails extend beyond the nucleosome, and
are sites of (mostly) reversible post-translational
modification
Other proteins wind
DNA into more tightly
packed form, the
chromosome.
Unwinding portions of
the chromosome is
important for mitosis,
replication and making
RNA.
H3 and H4 have been most extensively studied to date
Basically: The type of histone modification (mostly acetylation and
methylation) and the position of the modified amino acid determines
whether a gene will be expressed or not. Transcription factors and
associated proteins can modifiy the amino acids in the histone tails.
5
HAT
Ac
Ac
Ac
Ac
Bromodomain
Ac
• The action of Histone acetyl transferases (HATs) both decondenses
chromatin, and provides recognition sites for bromodomain containing
proteins (eg. Gcn5)
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 genome
contain a CpG island
- CpG are unmethylated
- But, when methylated, it leads to transcriptional repression
- Several tumors contained methylated CpG islands !!!
Example of a CpG island in the Retinoblastoma gene
An example of a CpG Island in the Retinoblastoma gene region. The
dotted line represents the statistically expected frequency of CpG sites
(1/16), while the solid line represents the measured frequency of CpG
sites in the 180 kb of DNA sequence that encompass the Rb gene exons
and introns. The location of two CpG islands is indicated by arrows. Only
the most 5' CpG island corresponds to the promoter of the gene.
http://www.mdanderson.org/departments/methylation/
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Transcription start, termination, RNA processing
in eukaryotes
DNA(gene)
promoter
Processing of pre-mRNAs
RNA polymerase II synthetised RNAs:
- capping (7m-Guanosine addition at 5’ of mRNA)
transcription
pre-mRNA
- poly-adenylation (50-200 Adenosines at 3’ of mRNA)
Cap
Splicing (Spliceosome)
mRNA
Cap
(A)n
translation
Protein
- Important difference between pro- and eukaryotes is that
genes in eukaryotes consist of exons and introns.
- Introns removed by a process called “splicing”
Messenger RNA 5' Cap
-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.
Example:
Mouse
Beta-Globin
Gene
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Nuclear Introns and splicing
(removal of intron sequences)
Nuclear mRNA Intron Removal
Rol van U-RNAs: herkenning van exon-intron grenzen en “branchpoint”
- 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
Intron Removal in Nuclear RNA
“Splicing”
Gene recognition in the genome
- Scan genomic DNA for exon/intron sequences, promoter
sequences, open reading frames, etc.
-Relevant informations comes from RNA because gene
sequences 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 complete
mRNAs, non-coding RNAs) (RNAs > cDNA)
- compare ESTs with genomic DNA
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Protein domains and exon-shuffling
hypothesis for the assembly and origin of
“new” genes.
Modular build-up of proteins: visualized
Many Proteins have a modular structure: functional domains
> Each domain has a specific function, and can be shared by
different proteins: Some proteins contain multiple copies of
a domain.
Examples:
Model: Assembly of different modules into a single protein
occurs via exon shuffling at the DNA level:
>> 1 or more exons encode for a particular protein domain; By DNA
rearrangements or via a RNA, exon sequences can be duplicated and
inserted in other genomic sites; for example, in other genes.
With this mechanism, it is assumed that new genes are created.
- 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.
Example
2 distinct
proteins
Calmodulin: Ca-binding
Both functions into
one protein
Senses [Ca++ ]
Kinase (activated by Calmodulin- Ca+ )
Model: by exon-shuffling DNAs combined
Not on scale: Principle!
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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
Gene/Protein Evolution
Definition of important concepts in (comparative) genomics
Homologs > paralogs + orthologs
- 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
- 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)
Homologs
•
> Paralog
–
•
Derived by duplication
> Ortholog
–
- Parologous genes or paralogs:
Common ancestor
Common 3D Structure
At least some sequence similarity
(sequence motifs or more close
similarity)
Derived by Speciation
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)
,
Anastasia Nikolskaya Georgetown
Univ.
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Craniata
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Am
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Te
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Myxi
Myo (Rat)
HbA (Rat)
HbB (Rat)
HbA (Frog)
HbB (Frog)
Myo (Frog)
HbA (Cod)
HbB (Cod)
Myo (Cod)
Hb (Hagfish)
Myo (Hagfish)
Orthologs and Paralogs
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Thanks for your attention !!
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Anastasia Nikolskaya Georgetown Univ
,
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