Basic Molecular Biology

Basic Molecular Biology

Structures of biomolecules How does DNA function? What is a gene? Computer scientists vs Biologists

Bioinformatics schematic of a cell

Macromolecule (Polymer)

Monomer

DNA Deoxyribonucleotides (dNTP)

RNA Ribonucleotides (NTP)

Protein or Polypeptide Amino Acid

Nucleic acids (DNA and RNA)

Form the genetic material of all living organisms.

Found mainly in the nucleus of a cell (hence “nucleic”)

Contain phosphoric acid as a component (hence “acid”)

They are made up of nucleotides.

Nucleotides

A nucleotide has 3 components Sugar (ribose in RNA, deoxyribose in DNA) Phosphoric acid Nitrogen base

Adenine (A) Guanine (G) Cytosine (C) Thymine (T) or Uracil (U)

Monomers of DNA

A deoxyribonucleotide has 3 components Sugar - Deoxyribose Phosphoric acid Nitrogen base

Adenine (A) Guanine (G) Cytosine (C) Thymine (T)

Monomers of RNA

A ribonucleotide has 3 components Sugar - Ribose Phosphoric acid Nitrogen base

Adenine (A) Guanine (G) Cytosine (C) Uracil (U)

Nucleotides

Phosphate Group

Sugar

NitrogenousBase

Phosphate Group

Sugar

NitrogenousBase

T

C

A

C

T

G

G

C

G

A

G

T

C

A

G

C

G

A

G

U

C

A

G

C

DNA RNA

A = T

G = C

T U

Composed of a chain of amino acids.

R

|

H2N--C--COOH

|

H

Proteins

20 possible groups

R R | | H2N--C--COOH H2N--C--COOH | | H H

Proteins

Dipeptide

R O R | II | H2N--C--C--NH--C--COOH | | H H

This is a peptide bond

Protein structure

Linear sequence of amino acids folds to form a complex 3-D structure.

The structure of a protein is intimately connected to its function.

Structure -> Function

It is the 3-D shape of proteins that gives them their working ability – generally speaking, the ability to bind with other molecules in very specific ways.

DNA: information store

RNA:information store and catalyst

Protein: superior catalyst

DNA in action

Questions about DNA as the carrier of genetic information: What is the information? How is the information stored in DNA? How is the stored information used ?

Answers: Information = gene → phenotype Information is stored as nucleotide sequences. .. and used in protein synthesis.

How does the series of chemical bases along a DNA strand (A/T/G/C) come to specify the series of amino acids making up the protein?

The need for an intermediary

Fact 1 : Ribosomes are the sites of protein synthesis.

Fact 2 : Ribosomes are found in the cytoplasm.

Question : How does information ‘flow’ from DNA to protein?

The Intermediary

Ribonucleic acid (RNA) is the “messenger”. The “messenger RNA” (mRNA) can be

synthesized on a DNA template. Information is copied (transcribed) from DNA

to mRNA. (TRANSCRIPTION)

Biological functions of RNA

• Mediate of the protein synthesis

• Messenger RNA (nRNA)

• Transfer RNA (tRNA)

• Ribosomal RNA (rRNA)

• Structural molecule: Ribosomal RNA

• Catalytic molecule: ribozyme

• Guide molecule: primer of DNA replication, protein degradation (tm RNA)…

• Ribonucleoprotein (complex of RNA and protein): mRAN edition, mRAN spicing, protein transport…

DNA

TRANSCRIPTION

rRNA mRNA tRNA

ribosome

TRADUCTION

PROTEINE

Transcription

The DNA is contained in the nucleus of the cell.

A stretch of it unwinds there, and its message (or sequence) is copied onto a molecule of mRNA.

The mRNA then exits from the cell nucleus. Its destination is a molecular workbench in

the cytoplasm, a structure called a ribosome.

Principal steps of the transcription

1. Polymerase RNA randomly binds on the DNA and seeks for a promoter (5’ 3’)

2. Opening of the DNA

3. Initiation of the polymerization

4. Elongation:

20-50 nucleotides/sec

1 error/104 nucleotides

5. Termination (at the termination signal)

RNA polymerase

It is the enzyme that brings about transcription by going down the line, pairing mRNA nucleotides with their DNA counterparts.

Promoters

Promoters are sequences in the DNA just upstream of transcripts that define the sites of initiation.

The role of the promoter is to attract RNA polymerase to the correct start site so transcription can be initiated.

5’Promoter 3’

Promoters

Promoters are sequences in the DNA just upstream of transcripts that define the sites of initiation.

The role of the promoter is to attract RNA polymerase to the correct start site so transcription can be initiated.

5’Promoter 3’

Promoter

So a promoter sequence is the site on a segment of DNA at which transcription of a gene begins – it is the binding site for RNA polymerase.

Termination site of the transcription

Next question…

How do I interpret the information carried by mRNA?

Think of the sequence as a sequence of “triplets”.

Think of AUGCCGGGAGUAUAG as AUG-CCG-GGA-GUA-UAG.

Each triplet (codon) maps to an amino acid.

Translation: mRNA protein

• Codons UAA, UAG and UGA are stop codons because there is no corresponding tRNA (except exception…);• Codon AUG code for initiator methionine (except exception); • The code is almost-universal.

The Genetic Code

Translation

At the ribosome, both the message (mRNA) and raw materials (amino acids) come together to make the product (a protein).

Translation

The sequence of codons is translated to a sequence of amino acids.

How do amino acids get to the ribosomes? They are brought there by a second type of RNA,

transfer RNA (tRNA).

Translation

Transfer RNA (tRNA) – a different type of RNA. Freely float in the cytoplasm. Every amino acid has its own type of tRNA that

binds to it alone. Anti-codon – codon binding crucial.

tRNA

tRNAOne end of the tRNA links with a specific amino acid, which it finds floating free in

the cytoplasm.

It employs its opposite end to form base pairs with

nucleic acids – with a codon on the mRNA tape that is

being read inside the ribosome.

tRNA

Transfer RNA

• 61 different tRAN, composed of from 75 to 95 nucleotides

• Recognition of a codon and binding to the corresponding amino acid

Elongation of the translation

The ribosome move by 3 nucleotides toward 3’ (elongation); in 1 second a Bacteria ribosome adds 20 amino acids!Eucaryote: 2 amino acids/second !

A stop codon stop (UAA, UAG, AGA) In the same reading frame, end the process; the ribosome break away from the mRNA.

Polyribosome (polysomes): eukaryote and prokaryote

Duration of the protein synthesis: between 20 seconds and several minutes: multiple initiations

~80 nucleotides between 2 ribosomes

Eukaryotes: 10 ribosomes / mRNAProcaryotes: up to 300 ribosomes / mRNA

The gene and the genome

A gene is a length of DNA that codes for a protein.

Genome = The entire DNA sequence within the nucleus.

Organism Sizee (bp) Number of genes

% coding

Remarks

E.coli 4,639,221 4,397

87 % Eubacterie

Methanococcus jannashii

1,664,970 1,758 87 % Archae

Saccharomyces cerevisiae

12,057,849 6,551 72 %

Arabidopsis thaliana

~135,000,000 ~ 25’000 ?

Caenorhabditis elegans

87,567,338 17,687

21 % 1000 cells

Drosophila melanogaster

~180,000,000 ~13,600 20 % Core proteome: 8,000 (families)

Human ~3,000,000,000 20,000-25,000

4-7 % (?)

Estimate of the number of genes (proteins + tRNA + rRNA)

Genome coding regions

Gene definition

• Nucleic acid sequence required for the synthesis of:

• a functional polypeptide

• a functional RNA (tRNA, rRNA,…)

• A gene coding for a protein generally contains:

• a coding sequence (CDS)

• control regions for transcription and translation (promoter, enhancer, poly A site…)

A gene contains coding and non-coding regions

More complexity

The RNA message is sometimes “edited”. Exons are nucleotide segments whose

codons will be expressed. Introns are intervening segments (genetic

gibberish) that are snipped out. Exons are spliced together to form mRNA.

Standard structure of a gene for vertebrate

RNA processing: Splicing

• Pre-messenger RNA contains coding sequence regions (exon: express sequence) alternate with non-coding regions (intron: intervening sequence)

• Splicing: excision of the introns

• High variability of the number of intron between genes in a given specieEx: human: from 2 introns (insulin) to more than 100 introns (117 introns collagen type VII)

• High variability of the number of intron between species : Ex: yeast gene has few introns (max 2 introns / gene).

• High variability of the size of the introns (min 18 nucleotides; to 300 kb);

• High variability of the size of the exons (min 8 coding nucleotides);

• Mitochondrial human genes do not contain introns, but mitochondrial vegetal and fungus (yeast include) contain introns; chloroplast’s genes contain introns; there exists introns for some prokaryotes !

• Importance in evolution; facilitate genetic recombination; linked with the notion of domains in proteins

• Human: average: 7kb intron / 1 kb exon;

Splicing: generalities

Alternative splicing

The exon order is generally fixed (except for exon scrambling)

Summery of the whole process

Proteins

• Several levels from primary to quaternary structure

• Composed of amino acids

0

1

2

3

4

5

6

7

8

9

10

% frequency

L A S G V E T K I R D P N Q F Y M H C W

Amino acid

Protein Structure

Proteins are poly-peptides of 70-3000 amino-acids

This structure is (mostly) determined by the sequence of amino-acids that make up the protein

Functional categories Enzymes Kinase, Protéase Transport Hemoglobin, Regulation Insuline, Répresseur lac Storage Caséine, Ovalbumine Structure Protéoglycan, Collagène Contraction Actine, Myosine Protection Immunoglobulines, Toxines Scaffold proteins Grb 2, crk Exotics Resiline, protéines adhésives

Number of proteins in various organisms

Organism Number

Bacteria 500-6’000Yeast 6’000C. elegans 19’000 Drosophila 15’000 Human 30’000-1’000’000

Protein Structure

Example of structural motif: HTH

• Helix – Turn – Helix (HTH) motif very common (prokaryotes et eukaryotes)

• DNA binding site for procaryotes:

From Genome to Proteome

ProteomeProteome

Alternative splicingof mRNA

Post-translational protein

modification (PTM)

Definition of PTM:Any modification of a polypeptide chain

that involves the formation or breakage ofa covalent bond.

Increase in complexity

10 -42 %5 to 10 fold

GenomeGenome

Human: about 25’000 genes

Human: about one million proteins; several proteomes

« After ribosomes »

Evolution

Related organisms have similar DNA Similarity in sequences of proteins Similarity in organization of genes along the

chromosomesEvolution plays a major role in biology

Many mechanisms are shared across a wide range of organisms

During the course of evolution existing components are adapted for new functions

Evolution

Evolution of new organisms is driven byDiversity

Different individuals carry different variants of the same basic blue print

Mutations The DNA sequence can be changed due to

single base changes, deletion/insertion of DNA segments, etc.

Selection bias

Numerous possible effect of mutation

Neutral basic Lys -> basic Arg

ADN 3’-AAA GCT ACC TAT CGG TCT 5’5’-TTT CGA TGG ATA GCC AGA 3’N-Phe Arg Trp Ile Ala Arg-C

Missense

ADN 3’-AAT GCT ACC TAT CGG TTT 5’5’-TTA CGA TGG ATA GCC AAA 3’N-Leu Arg Trp Ile Ala Lys-C

Nonsense

ADN 3’-AAA GCT ATC TAT CGG TTT 5’5’-TTT CGA TAG ATA GCC AAA 3’N-Phe Arg Stop

Frameshift (délétion de 4 bases)

ADN 3’-AAA CCT ATC GGT TT 5’5’-TTT GGA TAG CCA AA 3’N-Phe Gly Stop

Frameshift (insertion d’une base)

ADN 3’-AAA GCT ACC ATA TCG GTT T 5’5’-TTT CGA TGG TAT AGC CAA A 3’N-Phe Arg Trp Tyr Ser Gln

Original sequence

AminoAcids N-PheArg Trp Ile Ala Lys-C

ARNm 5’-UUU CGA UGG AUA GCC AAA-3’ADN 3’-AAA GCT ACC TAT CGG TTT 5’

5’-TTT CGA TGG ATA GCC AAA 3’

The Tree of Life

Sou

rce:

Alb

erts

et

al

Central dogma

DNA

tRNA

rRNA

snRNA

mRNA

transcription

translation

POLYPEPTIDE

ZOOM IN

Bioinformatics Studies the flow of information in biomedicine

Information flow from genotype to phenotypeDNA → Protein → Function → Organism → Population → DNA

Experimental flow for creating and testing modelsHypothesis → Experiment → Data → Conflict → Hypothesis

Computational Biology and Bioinformatics

The systematic development and application of computing systems and computational solution techniques to the analysis of biological data obtained by experiments, modeling, database search, and experimentation

Explosion of experimental data Difficulty in interpreting data Need for new paradigms for computing with data and

extracting new knowledge from it

Brief history of early bioinformatics• Molecular sequences and data bases

Dayhoff (atlas of proteins, 1965) Zuckerkandl & Pauling (1965), Bilofsky (GenBank, 1986), Hamm & Cameron (EMBL, 1986), Bairoch (Swiss-Prot, 1986)

• Molecular sequence comparison NeedleMan & Wunsch (1970), Smith & Waterman (1981), Pearson-Lipman (Fasta, 1985), Altschul (Blast, 1990)

• Multiple alignment and automatic phylogeny Aho (common subsequence, 1976), Felsenstein (infering phylogenies, 1981-1988), Sankoff & Cedergren (multiple comparison, 1983), Feng & Doolittle (Clustal, 1987), Gusfield (inferring evolutionary trees, 1991), Thompson (ClustalW, 1994)

• Motif search and discoveryFickett (ORF, 1982), Ukkonen (approximate string matching, 1985), Jonassen (Pratt, 1995), Califano (Splash, 2000) Pevzner (WINNOVER, 2000)

• But also: RNA structure prediction, protein threading, protein foldings… Few fields and large use of combinatoric/dynamic

programming approaches

New biological data imply new bioinformatics field • Sequence

Motif search, motif discovery, alignment…

Data indexing, regular language, dynamic programming, HMM, EM, Gibbs sampling…

• Structure

RNA folding, protein threading, protein folding…

Palindrome search, context-(free, sensitive) language, dynamic programming, combinatorial optimization…

• DNA chip

Classification, clustering, feature selection, regulation network…

NN, SVM, Bayesian inference, (hierarchical, k, Gaussian)-clustering, differencial model…

• Proteomics

Spectrum analysis, image pattern matching, probabilistic model…

• Bibliographic data

Ontology, text mining…

Important source of data and information

GENEBANK: http://www.ncbi.nih.gov

Swiss-prot: http://us.expasy.org/sprot/relnotes

Protein Data Bank (PDB): http://www.rcsb.org/pdb/home/home.do

Stanford Microarray DB http://smd.stanford.edu

MedLine or PubMed http://genome.ucsc.edu or http://www.ebi.ac.uk/ensembl

Journals: Bioinformatics, BMC bioinformatics, Nucleic Acids Research, Journal of Molecular Biology, Proteomics…

Computer scientists vs Biologists

(Almost) Nothing is ever completely true or false in Biology.

Everything is either true or false in computer science.

Computer scientists vs Biologists

Biologists strive to understand the very complicated, very messy natural world.

Computer scientists seek to build their own clean and organized virtual worlds.

Computer scientists vs Biologists

Biologists are more data driven. Computer scientists are more algorithm

driven. One consequence is CS www pages have

fancier graphics while Biology www pages have more content.

Computer scientists vs Biologists

Biologists are obsessed with being the first to discover something.

Computer scientists are obsessed with being the first to invent or prove something.

Computer scientists vs Biologists

Biologists are comfortable with the idea that all data has errors.

Computer scientists are not.

Computer scientists vs Biologists

Computer scientists get high-paid jobs after graduation.

Biologists typically have to complete one or more post-docs...

Computer Science is to Biology what Mathematics

is to Physics