1

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

2

RNA Polymerases Where does RNA pol bind?

Pribnow Box of Promoter: ProkaryoteTemplate (Anticoding) Strand

>> terminologie

3

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’

4

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)

5

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.

6

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/

7

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

8

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

9

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.

10

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

11

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 !!